Methods and products for in vivo enzyme profiling

ABSTRACT

The present invention relates to methods and products associated with in vivo enzyme profiling. In particular, the invention relates to methods of in vivo processing of exogenous molecules followed by detection of signature molecules as representative of the presence of active enzymes associated with diseases or conditions. The invention also relates to products, kits, and databases for use in the methods of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/091,075, filed Nov. 6, 2020, entitled “METHODS AND PRODUCTS FOR INVIVO ENZYME PROFILING”, which is a continuation of U.S. application Ser.No. 15/966,385, filed Apr. 30, 2018, entitled “METHODS AND PRODUCTS FORIN VIVO ENZYME PROFILING”, now issued as U.S. Pat. No. 10,883,998 onJan. 5, 2021, which is a divisional of U.S. application Ser. No.14/166,481, filed Jan. 28, 2014, entitled “METHODS AND PRODUCTS FOR INVIVO ENZYME PROFILING”, now issued as U.S. Pat. No. 9,970,941 on May 15,2018, which is a continuation of U.S. application Ser. No. 12/715,965,filed on Mar. 2, 2010, entitled “METHODS AND PRODUCTS FOR IN VIVO ENZYMEPROFILING,” now issued as U.S. Pat. No. 8,673,267 on Mar. 18, 2014,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/156,660, filed on Mar. 2, 2009, entitled “METHODS ANDPRODUCTS FOR IN VIVO ENZYME PROFILING,” the entire contents of each ofwhich are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 7, 2022, isnamed M065670181US05-SEQ-FL.txt and is 14,982 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under CA124427 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to methods and products associated with invivo enzyme profiling. Some aspects of the present invention relate toprofiling enzymatic reaction products. In particular, the inventionrelates to methods of in vivo processing of exogenous molecules followedby detection of signature molecules as representative of the presence orabsence of active enzymes associated with disease or conditions. Theinvention also relates to products, kits, and databases for use in themethods of the invention.

BACKGROUND OF THE INVENTION

Dysregulation of proteases in cancer has important consequences in cellsignaling and helps drive cancer cell proliferation, invasion,angiogenesis, avoidance of apoptosis, and metastasis. Currently, in vivoanalysis of proteases (and other enzymes such as glycosidase, esterase,etc.) activity is limited to biopsy or local fluorescent techniques,which are hindered by their invasiveness or low multiplexing potential,respectively.

SUMMARY OF THE INVENTION

The invention in some aspects is a method involving administering to asubject a pro-diagnostic reagent, wherein the pro-diagnostic reagentcomprises a modular structure having a carrier domain linked to asignature producing domain, wherein the signature producing domain iscapable of producing a signature molecule in the subject; identifying abiological sample for detection of the signature molecule, wherein thebiological sample is at a site remote from the production of thesignature molecule; and, subjecting the biological sample to an analysismethod in order to detect the presence of the signature molecule,wherein the presence of the signature molecule in the biological sampleis indicative of a biological predictor molecule within the subject.

In one embodiment the signature producing domain comprises an enzymesusceptible domain linked to a signature molecule, wherein thebiological predictor molecule is an enzyme, wherein the enzymesusceptible domain is susceptible to modification by the enzyme in thesubject, and wherein the presence of the signature molecule in thebiological sample is indicative of an active enzyme within the subject.

In another embodiment the signature producing domain comprises an activesignature producing agent, wherein the active signature producing agentis capable of modifying the biological predictor molecule to produce thesignature molecule in the subject. The active signature producing agentmay be an enzyme, such as a protease or a glycosidase.

In other embodiments the pro-diagnostic reagent further comprises animplantable microdelivery device that houses the modular structure. Theimplantable microdelivery device in some embodiments is an implantablecapsule with a semi-permeable membrane that encapsulates the modularstructure. In other embodiments the implantable microdelivery device isa chip having the modular structure attached thereto. In yet otherembodiments the implantable microdelivery device is a sustained-releaseformulation.

In some aspects the invention is a method involving administering to asubject a pro-diagnostic reagent, wherein the pro-diagnostic reagentcomprises a carrier domain linked to an enzyme susceptible domain whichis linked to a signature molecule, wherein the enzyme susceptible domainis susceptible to modification by an enzyme in the subject; identifyinga biological sample for detection of the signature molecule, wherein thebiological sample is at a site remote from the enzyme; and, subjectingthe biological sample to an analysis method in order to detect thepresence of one or more signature molecules, wherein the presence of thesignature molecule in the biological sample is indicative of an activeenzyme within the subject.

In other aspects of the invention a method of administering to a subjecta pro-diagnostic reagent, wherein the pro-diagnostic reagent comprises acarrier domain linked to an enzyme susceptible domain which is linked toa signature molecule; collecting a urine sample from the subject; and,subjecting the urine sample to an analysis method in order to detect thepresence of the signature molecule, wherein the presence of thesignature molecule in the biological sample is indicative of an activeenzyme within the subject is provided.

In yet other aspects a method for diagnosing a disease is provided. Themethod involves administering to a subject a pro-diagnostic reagent,wherein the pro-diagnostic reagent comprises a carrier domain linked toan enzyme susceptible domain which is linked to a signature molecule,and wherein the enzyme susceptible domain is susceptible to cleavage byan enzyme associated with a disease; collecting a urine sample from thesubject; and, subjecting the urine sample to an analysis method in orderto detect the presence of the signature molecule, wherein the presenceof the signature molecule in the biological sample is indicative of thesubject having the disease.

In another aspect of the invention a method of collecting a urine samplefrom a subject suspected of having a disorder or condition, wherein thesubject has been administered a pro-diagnostic reagent, thepro-diagnostic reagent comprising a carrier domain linked to an enzymesusceptible domain which is linked to a signature molecule; and,subjecting the urine sample to a multiplex analysis method in order todetect the presence of the signature molecule, wherein the presence ofthe signature molecule in the biological sample is indicative of thedisorder or condition within the subject. is provided In someembodiments, the subject is a healthy subject. In some embodiments, thesubject is a subject at risk of developing a disease or condition. Insome embodiments, the subject is suspected of having a disease orcondition or a subject diagnosed with having a disease or condition.

A method of collecting a urine sample from a subject suspected of havinga disorder or condition, wherein the subject has been administered apro-diagnostic reagent, the pro-diagnostic reagent comprising a carrierdomain linked to an enzyme susceptible domain which is linked to asignature molecule and, subjecting the urine sample to a multiplexanalysis method in order to detect the presence or absence of thesignature molecule, wherein the absence of the signature molecule in thebiological sample is indicative of the disorder or condition within thesubject is provided according to other aspects of the invention.

A method of treating a subject is provided according to an aspect of theinvention. The method involves collecting a urine sample from a subjectsuspected of having a disorder or condition or diagnosed with a disorderor condition, wherein the subject has been administered a pro-diagnosticreagent, the pro-diagnostic reagent comprising a carrier domain linkedto an enzyme susceptible domain which is linked to a signature molecule;subjecting the urine sample to a multiplex analysis method in order todetect the presence of the signature molecule, wherein the presence ofthe signature molecule in the biological sample is indicative of thedisorder or condition within the subject; and, administering atherapeutic agent to the subject to treat the disorder.

In some embodiments a further step of collecting a biological samplefrom the subject is provided. In other embodiments the signaturemolecule is detected in the biological sample in the subject. Thebiological sample may be urine, blood, saliva, or mucous secretion.

A plurality of pro-diagnostic reagents having a plurality of signaturemolecules may be administered to the subject in some embodiments. Theplurality of pro-diagnostic reagents may have a plurality of signaturemolecules. In other embodiments the pro-diagnostic reagent includes aplurality of signature molecules.

In some embodiments the enzyme susceptible domain is susceptible tomodification, i.e. cleavage, addition, conformational or charge change,by an enzyme associated with a disease or condition. In some embodimentsthe enzyme susceptible domain is susceptible to cleavage by a proteaseassociated with a disease or condition. The enzyme susceptible domain inother embodiments is susceptible to modification by an enzyme notassociated with a disease or condition, but associated with a normalcondition.

In some embodiments the enzyme susceptible domain is a peptide, such as,for instance, a MMP sensitive site, a kallikrein sensitive site, acathepsin sensitive site, a plasminogen activator sensitive site and/oran ADAM sensitive site.

In some embodiments the disease or condition is cancer, cardiovasculardisease, arthritis, viral, bacterial, parasitic or fungal infection,Alzheimer's disease emphysema, thrombosis, hemophilia, stroke, organdysfunction, any inflammatory condition, vascular disease, parenchymaldisease, or a pharmacologically-induced state.

In some embodiments, the carrier domain comprises a particle, forexample, a microparticle or a nanoparticle. The carrier domain isgreater than 5 nm in size in some embodiments and in other embodimentsis smaller than 5 nm in size. In some embodiments, the carrier domaincomprises a targeting domain and/or a therapeutic agent. In someembodiments, the carrier domain selectively interacts with a moleculartarget, for example, a protein or peptide, a nucleic acid, or acarbohydrate. In some embodiments, the carrier domain selectively bindsa molecular target. In some embodiments, the carrier domain selectivelyinteracts with a target molecule as part of an enzymatic reaction, forexample, an enzymatic reaction carried out by the carrier domain or bythe target molecule. In some embodiments, the carrier domain comprises apeptide, a protein, a nucleic acid or a small molecule, for example, apeptide, protein, nucleic acid or small molecule selectively binding amolecular target, for example, a target molecule (e.g., a peptide,protein, nucleic acid, or carbohydrate) expressed in a target cell orcell type, after administration to a subject. In some embodiments themolecular target is specifically expressed in a target cell or targetcell type, for example, a cancer cell or a cell of a certaindifferentiation state or of a certain tissue. In some embodiments, thecarrier domain comprises a therapeutic agent. In some embodiments, thecarrier domain comprises a therapeutic agent selectively interactingwith a molecular target, for example, a molecular target expressed in atarget cell or target cell type. In some embodiments the carrier domainis a nanoparticle, a peptide, for example, an RGD peptide, an aptamer,an antibody, or a fragment thereof, an adnectin, or a targetingmolecule.

The signature molecule in some embodiments is a peptide, nucleic acid,small molecule, fluorophore/quencher, carbohydrate, particle,radiolabel, MRI-active compound, inorganic material, and/or organicmaterial, with encoded characteristics to facilitate optimal detection.

The analysis step used in the methods may be a multiplex analysis methodor a singular analysis method. The analysis methods include but are notlimited to mass spectrometry, liquid chromatography-mass spectrometry,PCR analysis, DNA microarray, and fluorescence analysis.

In some embodiments the method also includes a purification step,wherein the signature molecule is isolated from other components in thebiological sample. The purification step may be, for instance, affinitychromatography.

In other aspects of the invention a reagent is provided. The reagentincludes a carrier domain, wherein the carrier domain is a particle andis greater than 5 nm in size; an enzyme susceptible domain linked to thecarrier domain; and, a signature molecule linked to the enzymesusceptible domain, wherein the signature molecule is a peptide ornucleic acid.

In other aspects, the invention is a reagent having an implantablemicrodelivery device housing a modular structure having a carrier domainlinked to a signature producing domain.

In some embodiments the signature producing domain comprises an activesignature producing agent, wherein the active signature producing agentis capable of modifying a biological predictor molecule to produce asignature molecule. The active signature producing agent may be anenzyme such as a protease or a glycosidase. In other embodiments theimplantable microdelivery device is an implantable capsule with asemi-permeable membrane that encapsulates the modular structure, a chiphaving the modular structure attached thereto, or a sustained-releaseformulation.

In other aspects the invention is a reagent including a carrier domainhaving a plurality of enzyme susceptible domains linked to the carrierdomain wherein each enzyme susceptible domain is linked to anon-fluorescent signature molecule.

In yet other aspects the invention is a composition having a pluralityof pro-diagnostic reagents comprising a carrier domain, an enzymesusceptible domain linked to the carrier domain; and, a non-fluorescentsignature molecule linked to the enzyme susceptible domain.

In some embodiments the carrier domain is polymer based microparticle,an iron oxide microparticle, or nanoparticle, an inorganic carrier, oran organic carrier. The carrier domain optionally includes a targetingdomain and/or a therapeutic agent. The targeting domain may be, forinstance, an antibody.

In some embodiments the enzyme susceptible domain is a peptide, such asfor instance, GGPQGIWGQC (SEQ ID NO: 1), GGPLGVRGKC (SEQ ID NO: 2),GGPLANvaDpaARGC (SEQ ID NO: 3), GGPVGLIGL (SEQ ID NO: 4), GGPVPLSLVMC(SEQ ID NO: 5), GGSGGPLGLRSWC (SEQ ID NO: 6), GGGPWGIWGQGC (SEQ ID NO:7), GGdFPipRSGGGC (SEQ ID NO: 8), or GGLVPRGSGC (SEQ ID NO: 9).

In other embodiments the signature molecule is a peptide, nucleic acid,small molecule, fluorophore/quencher, carbohydrate, or particle. Thesignature molecule in some embodiments is a peptide of GGPQG (SEQ ID NO:10), GGPLG (SEQ ID NO: 11), GGPLA (SEQ ID NO: 12), GGPVG (SEQ ID NO:13), GGPVPLS (SEQ ID NO: 14), GGSGGPLG (SEQ ID NO: 15), GGGPWG (SEQ IDNO: 16), GGdFPipR (SEQ ID NO: 17), or GGLVP (SEQ ID NO: 18).

A kit is provided according to other aspects of the invention. The kithas a container housing a pro-diagnostic reagent, wherein thepro-diagnostic reagent comprises a carrier domain linked to an enzymesusceptible domain which is linked to a signature molecule; and,instructions for administering the pro-diagnostic reagent to a subjectand for analyzing the signature molecule of the pro-diagnostic reagentin a biological sample of the subject.

In some embodiments the kit also includes a second container housing ananalytical reagent. In other embodiments the kit also includes a boxhousing the containers. In yet other embodiments the kit includes aspecimen collection device. Other embodiments of this invention woulduse a diversity of carriers, cleavage domains, and signature moleculesto enable detection via modalities such as radiation, fluorescence,color, elemental detection, light scattering, magnetic techniques, MRI,electrical measurements, biochemical measurements, biological assays(including ELISA assays and others), among others.

Each of the embodiments of the invention can encompass variousrecitations made herein. It is, therefore, anticipated that each of therecitations of the invention involving any one element or combinationsof elements can, optionally, be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are s a schematic depicting a method according to theinvention for multiplexed in vivo enzyme profiling of mass-codednanoparticle based pro-diagnostic reagents.

FIGS. 2A-2E show data depicting the process of tumor and wound targetingwith pro-diagnostic reagents. FIG. 2A is a schematic of thepro-diagnostic reagent, with the circles referring to the carrier, thestar is a fluorescent molecule, and the zigzag line refers to the enzymesusceptible domain and the signature molecule (darker end region). FIG.2B is an electron micrograph of the pro-diagnostic reagent. FIG. 2C is agraph depicting the circulation time of the pro-diagnostic reagent, byplotting detection of the carrier in the blood with respect to timeafter intravenous injection. FIG. 2D is photographs of mice havingeither tumors or injuries (left and right panels, respectively)administered the pro-diagnostic reagent. FIG. 2E is histopathologicalanalysis of carrier homing to tumors or regions of injury.

FIG. 3A is a schematic of the pro-diagnostic reagent, with the circlesreferring to the carrier, the star is a signature molecule, and thezigzag line refers to the enzyme susceptible domain. FIG. 3B is a graphdepicting fluorescence activation versus time. FIG. 3C depicts data on43 pro-diagnostic reagents (with enzyme susceptible domains listed tothe right for detection of tumor and injury enzymes.

FIG. 4 is a Table depicting the mass detection of ejected fragments invitro. The results confirmed that the fluorescent results from thescreen could also be detected by analyzing the mass of ejected fragmentsin vitro.

FIGS. 5A-5C depict the results of fluorescent detection of urinaryreporter activation by tumors and injuries in vivo. FIG. 5A is aschematic of the pro-diagnostic reagent as shown in FIG. 3A, furtherdepicting the portion of the molecule that undergoes renal clearance andthe portion that undergoes RES clearance. FIG. 5B is a set ofphotographs of that were intravenously administered the optimizedpro-diagnostic reagent for injury detection (top) or tumor detection(bottom). Half the mice that were administered the optimizedpro-diagnostic agents for injury-detection suffered bilateral hind limbinjuries (left side of the photograph) while the control mice had noinjuries (right side of the photograph). Half the mice administered theoptimized pro-diagnostic agents for tumor-detection harbored humanfibrosarcoma tumors (HT-1080) (left side of photograph), while the othermice contained no tumors (right side of photograph). FIG. 5C is a set ofgraphs depicting relative bladder fluorescence for tumor (bottom panel)or injured (top panel) versus control mice in order to track theentrance of cleaved signature peptide into the urine after injection.

FIGS. 6A-6B show LC/MS quantitation of signature molecules in urine.FIG. 6A is a photograph of an experimental mouse, having bilateralinjury and a control uninjured mouse. FIG. 6B is a graph depicting theratio of signature molecule (from thrombin cleavable proteolyticsusceptible domain) to isotopically labeled product in injured versuscontrol mice.

FIG. 7A shows a photograph of a typical implantable capsule incomparison to a penny and a ruler. FIG. 7B is a graph showingmeasurements of thrombin-cleaved peptide efflux from implantablediagnostic capsules sealed with semi-permeable membranes of differentpore sizes. Capsules made with membranes of pore size 10, 30, 50 or 80nm were loaded with nanoparticles functionalized with GGdFPipRSGGGC (SEQID NO: 8) and exposed to solutions of thrombin or factor Xa (a cognateand a non-cognate protease, respectively). Thrombin-specific cleavagewas monitored over time and is shown in terms of kinetics of reporterrelease.

FIG. 8A shows representative proteolytic products appended with peptidecaps of differing length and charge density. FIG. 8B is a graph showingnormalized relative intensities of the peptide reporters. The inset ofFIG. 8B shows a magnification of the normalized relative intensities ofthe peptide sequences A1-A6 as measured via LC/MS.

FIG. 9A is a schematic of the pro-diagnostic reagent, showing twoidentical cocktails of 12 pro-diagnostic nanoparticles, eachfunctionalized with a different peptide, with the circles referring tothe carrier, the star is a signature molecule, and the zigzag linerefers to the enzyme susceptible domain. FIG. 9B is a graph depictingLC/MS peak area measurements of all twelve peptides after exposure ofthe first multiplex cocktail to thrombin. Left to right the sequences onthe X-axis correspond to SEQ ID NOs. 37-48. FIG. 9C is a graph depictingLC/MS peak area measurements of all twelve peptides after exposure ofthe second multiplex cocktail to collagenase. Left to right thesequences on the X-axis correspond to SEQ ID NOs. 37-48. FIG. 9D is agraph showing the ratio of the LC/MS peak area for each peptide reporterafter exposure to thrombin over the peak area measured after exposure tocollagenase. Left to right the sequences on the X-axis correspond to SEQID NOs. 49, 38, 39, 50, 41, 47, 51, 44, 45, 48, 46, and 42.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is GGPQGIWGQCSEQ ID NO: 2 is GGPLGVRGKC SEQ ID NO: 3 is GGPLANvaDapARGCSEQ ID NO: 4 is GGPVGLIGL SEQ ID NO: 5 is GGPVPLSLVMC SEQ ID NO: 6 isGGSGGPLGLRSWC SEQ ID NO: 7 is GGGPWGIWGQGC SEQ ID NO: 8 is GGdFPipRSGGGCSEQ ID NO: 9 is GGLVPRGSGC SEQ ID NO: 10 is GGPQG SEQ ID NO: 11 is GGPLGSEQ ID NO: 12 is GGPLA SEQ ID NO: 13 is GGPVG SEQ ID NO: 14 is GGPVPLSSEQ ID NO: 15 is GGSGGPLG SEQ ID NO: 16 is GGGPWG SEQ ID NO: 17 isGGdFPipR SEQ ID NO: 18 is GGLVP SEQ ID NO: 19 is GGVVVLSSEQ ID NO: 20 is Fl-dR-dS-dR SEQ ID NO: 21 is Fl-dR-G-dS-dRSEQ ID NO: 22 is Fl-dR-dS-dR-G-G-P-Q-G-I-W-G-Q-C SEQ ID NO: 23 isFl-dR-G-dS-dR-G-G-P-L-G-V-R-G-K-C SEQ ID NO: 24 isFl-dR-G-dS-dR-G-G-P-L-A-Nva-Dpa-A-R-G-C SEQ ID NO: 25 isFl-dR-G-dS-dR-G-G-P-V-G-L-I-G-C SEQ ID NO: 26 isFl-dR-dS-dR-G-G-P-V-P-L-S-L-V-M-C SEQ ID NO: 27 isFl-dR-G-dS-dR-G-G-V-V-V-L-S-M-T-A-C SEQ ID NO: 28 isFl-dR-G-dS-dR-G-G-S-G-G-P-L-G-L-R-S-W-C SEQ ID NO: 29 isFl-dR-G-dS-dR-G-G-G-P-W-G-I-W-G-Q-G-C SEQ ID NO: 30 isFl-dR-G-G-dS-G-G-dF-Pip-R-S-G-G-G-C SEQ ID NO: 31 isFl-dR-dS-dR-G-G-L-V-P-R-G-S-G-C SEQ ID NO: 32 isFl-dR-G-G-dS-G-G-F-P-R-S-G-G-G-C SEQ ID NO: 33 isFl-dR-G-G-dS-G-G-G-dF-Pip-K-S-G-G-G-C SEQ ID NO: 34 isFl-dR-G-G-dS-G-G-G-dF-P-K-S-G-G-G-C SEQ ID NO: 35 is dR-dS-dRSEQ ID NO: 36 is dR-G-dS-dR(Fl: Fluorescein; Nva: Norvaline; Dap = (N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid); Pip: pipecolic acid; d: D-isomer.)

DETAILED DESCRIPTION OF THE INVENTION

The status of physiological conditions of a subject can be assessedusing the methods of the invention by identifying molecular propertiesalso referred to as “molecular signatures”. Such molecular signaturesare useful for diagnosing diseases such as cancer, rheumatoid arthritisand arteriosclerosis, as well as for prognostic indicators. The responseof most cancers to medical intervention is currently monitored byphysical exams and various clinical imaging modalities. A few cancerssuch as prostate and ovarian cancer are monitored by use of singlebiomarkers in the blood. Such diagnostic techniques are achieved, forinstance using fluorescence detection of molecular markers which areactivated in a particular disease state.

The invention relates to a platform for functional characterization ofdisease or condition specific enzymatic repertoire as a method tomonitor both disease progression and regression as well as response totherapeutics. The methods provide orders of magnitude more in vivoenzyme-substrate information than current fluorescent detectiontechnologies. The platform provides a unique opportunity to functionallymonitor cancer and other disease progression and response to therapy. Itis particularly useful for prolonged therapeutic regimens, where thediscovery of prognostic functional signatures would greatly assistintervention and where enzymatic signatures directly correlate totherapeutic efficacy.

By administering a pro-diagnostic reagent, such as an exogenousdetectable substrate library into animal models of disease it ispossible to gain information into substrate specific enzymaticactivities associated with diseases, such as cancer, cardiovasculardisease, arthritis, and others. The technology allows for the potentialsimultaneous profiling of hundreds of enzyme-substrate activities invivo using, for instance, -chaperoned, enzyme sensitive detectablecompounds, an example of a compound referred to as pro-diagnosticreagents. The method leverages the distinct pharmacokinetics of modularstructures and small, optionally hydrophilic, marker peptides (RES andrenal clearance, respectively). The pro-diagnostic reagents have longcirculation times and thus remain in circulation or permeate into tumorsvia porous angiogenic vascular networks, where upon local molecules,such as enzymes (MMPs, kallikreins, cathepsins, plasminogen activators,ADAMs) gain access to the enzyme susceptible regions of thepro-diagnostic reagents or substrates gain access to the enzymes of thepro-diagnostic reagents.

When the pro-diagnostic reagents, for example, the reagents including anenzyme susceptible domain are exposed to enzymes, for instance,proteases, the reagent is cleaved, such that a marker, referred toherein as a signature molecule, is released. The marker isrenally-cleared and thus functions as a “messenger” of enzyme activity.For instance, a marker may include a self-quenched dye, such as Cy5.5which is bound to a larger molecule. When the peptide containing theself-quenched dye is cleaved or modified by specific enzymes at thedisease site the fluorophores are no longer self-quenched but insteaddeveloped fluorescent properties which can be detected at remote sites.Alternatively, using mass-encoded substrate libraries, the mass ofenzyme substrates are designed such that upon cleavage, a distinctmass-specific messenger of cleavage will enter the urine of a patient oranimal for detection using LC-MS technology. LC-MS urine analysis cangenerate data that is organized into a barcode of, for instance, cancerenzyme activity. In the absence of enzyme activity the pro-diagnosticreagents remain uncleaved and the whole reagent including the signaturemolecule is cleared through RES organs (liver, spleen, and lymph nodes)without producing urine markers. The use of mass to identify substratesallows unprecedented multiplexing capability with the potential to assaygreater than 1,000 substrates.

When the pro-diagnostic reagents includes an enzyme, such as a protease,the enzyme is exposed to endogenous substrates and the substrate iscleaved, such that a marker, referred to herein as a signature molecule,is released from the endogenous substrate. The marker is renally-clearedand thus functions as a “messenger” of enzyme activity. For instance, amarker may include a peptide, carbohydrate or nucleic acid fragmentwhich has been cleaved from the substrate. Using the detectiontechniques, for instance, LC-MS technology, the mass of the signaturecan be detected. LC-MS urine analysis can generate data that isorganized into a barcode of, for instance, cancer enzyme/substrateactivity. In the absence of enzyme activity the signature molecule isnot cleared through RES organs (liver, spleen, and lymph nodes) and doesnot produce urine markers.

Thus, the invention in some aspects involves administering to a subjecta pro-diagnostic reagent, identifying a biological sample from thesubject in which to detect the signature molecule and optionallycollecting the sample; and, subjecting the biological sample to ananalysis method in order to detect the presence of one or more signaturemolecules. The presence of the signature molecule in the biologicalsample is indicative of an active enzyme or a substrate within thesubject.

For example the invention in some aspects involves methods foradministering to a subject a pro-diagnostic reagent, such that thepro-diagnostic reagent has a modular structure having a carrier domainlinked to a signature producing domain, wherein the signature producingdomain is capable of producing a signature molecule in the subject;identifying a biological sample for detection of the signature molecule,wherein the biological sample is at a site remote from the production ofthe signature molecule; and, subjecting the biological sample to ananalysis method in order to detect the presence of the signaturemolecule, wherein the presence of the signature molecule in thebiological sample is indicative of a biological predictor moleculewithin the subject.

The pro-diagnostic reagent comprises a modular structure having acarrier domain linked to a signature producing domain. A modularstructure, as used herein, refers to a molecule having multiple domains.

The signature producing domain may be, for instance, an enzyme that canreact with an endogenous substrate in a subject to produce a signaturemolecule or it may be an enzyme susceptible domain which is linked to asignature molecule. The carrier domain may include a single type ofsignature producing domain, such as, a single enzyme susceptible domainand or signature molecule or it may include multiple signature producingdomains, such as, different enzyme susceptible domains and signaturemolecules. For instance each carrier may include 1 type of signatureproducing domain or it may include 2-1,000 different signature producingdomains or any integer therebetween. Alternatively each carrier mayinclude greater than 1,000 signature producing domains. Multiple copiesof the pro-diagnostic reagent are administered to the subject. Somemixtures of pro-diagnostic reagents may include signature producingdomains that are enzymes, others may be enzymatic susceptible domains,and other may be mixtures of the two. Additionally a plurality ofdifferent pro-diagnostic reagents may be administered to the subject todetermine whether multiple enzymes and/or substrates are present. Inthat instance, the plurality of different pro-diagnostic reagentsincludes a plurality of signature molecules, such that each enzymesusceptible domain is associated with a particular signature molecule ormolecules.

The carrier domain may serve as the core of the pro-diagnostic agent. Apurpose of the carrier domain is to serve as a platform for thesignature producing domain. As such, the carrier can be any material orsize as long as it can serve as a carrier or platform. Preferably thematerial is non-immunogenic, i.e. does not provoke an immune response inthe body of the subject to which it will be administered. Anotherpurpose is that it may function as a targeting means to target themodular structure to a tissue, cell or molecule. In some embodiments thecarrier domain is a particle. A particle, for example, a nanoparticle,may, for instance, result in passive targeting to tumors by circulation.Other types of carriers, include, for instance, compounds that causeactive targeting to tissue, cells or molecules. Examples of carriersinclude, but are not limited to, microparticles, nanoparticles,aptamers, peptides (RGD, iRGD, LyP-1, CREKA, etc.) antibodies orantibody fragments (e.g. herceptin, cetuximab, panitumumab, etc.) andsmall molecules (e.g. erlotinib, gefitinib, sorafenib, etc.).

As used herein the term “particle” includes nanoparticles as well asmicroparticles. Nanoparticles are defined as particles of less than 1.0μm in diameter. A preparation of nanoparticles includes particles havingan average particle size of less than 1.0 μm in diameter. Microparticlesare particles of greater than 1.0 μm in diameter but less than 1 mm. Apreparation of microparticles includes particles having an averageparticle size of greater than 1.0 μm in diameter. The microparticles maytherefore have a diameter of at least 5, at least 10, at least 25, atleast 50, or at least 75 microns, including sizes in ranges of 5-10microns, 5-15 microns, 5-20 microns, 5-30 microns, 5-40 microns, or 5-50microns. A composition of particles may have heterogeneous sizedistributions ranging from 10 nm to mm sizes. In some embodiments thediameter is about 5 nm to about 500 nm. In other embodiments, thediameter is about 100 nm to about 200 nm. In other embodiment, thediameter is about 10 nm to about 100 nm.

The particles may be composed of a variety of materials includingceramic, metallic, natural polymer materials (including lipids, sugars,chitosan, hyaluronic acid etc), synthetic polymer materials (includingpoly-lactide-coglycolide, poly-glycerol sebacate, etc), and non-polymermaterials, or combinations thereof.

The particles may be composed in whole or in part of polymers ornon-polymer materials. Non-polymer materials, for example, may beemployed in the preparation of the particles. Exemplary materialsinclude alumina, calcium carbonate, calcium sulfate, calciumphosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate,hydroxyapatite, tricalcium phosphate, dicalcium phosphate, tricalciumphosphate, tetracalcium phosphate, amorphous calcium phosphate,octacalcium phosphate, and silicates. In certain embodiments theparticles may comprise a calcium salt such as calcium carbonate, azirconium salt such as zirconium dioxide, a zinc salt such as zincoxide, a magnesium salt such as magnesium silicate, a silicon salt suchas silicon dioxide or a titanium salt such as titanium oxide or titaniumdioxide.

A number of biodegradable and non-biodegradable biocompatible polymersare known in the field of polymeric biomaterials, controlled drugrelease and tissue engineering (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404 to Vacanti; U.S.Pat. Nos. 6,095,148; 5,837,752 to Shastri; U.S. Pat. No. 5,902,599 toAnseth; U.S. Pat. Nos. 5,696,175; 5,514,378; 5,512,600 to Mikos; U.S.Pat. No. 5,399,665 to Barrera; U.S. Pat. No. 5,019,379 to Domb; U.S.Pat. No. 5,010,167 to Ron; U.S. Pat. No. 4,946,929 to d′Amore; and U.S.Pat. Nos. 4,806,621; 4,638,045 to Kohn; see also Langer, Acc. Chem. Res.33:94, 2000; Langer, J. Control Release 62:7, 1999; and Uhrich et al.,Chem. Rev. 99:3181, 1999; all of which are incorporated herein byreference).

Polymers include, but are not limited to: polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyglycolides, polysiloxanes, polyurethanes andcopolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexlmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride and polystyrene.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide)and poly(lactide-co-caprolactone), and natural polymers such asalgninate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof (substitutions, additions ofchemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art), albumin and other hydrophilic proteins, zein and otherprolamines and hydrophobic proteins, copolymers and mixtures thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion. The foregoingmaterials may be used alone, as physical mixtures (blends), or asco-polymers. In some embodiments the polymers are polyesters,polyanhydrides, polystyrenes, polylactic acid, polyglycolic acid, andcopolymers of lactic and glycoloic acid and blends thereof.

PVP is a non-ionogenic, hydrophilic polymer having a mean molecularweight ranging from approximately 10,000 to 700,000 and the chemicalformula (C6H₉NO)[n]. PVP is also known aspoly[1-(2-oxo-1-pyrrolidinyl)ethylene], Povidone™, Polyvidone™, RP 143™,Kollidon™, Peregal ST™, Periston™, Plasdone™, Plasmosan™, Protagent™Subtosan™, and Vinisil™. PVP is non-toxic, highly hygroscopic andreadily dissolves in water or organic solvents.

Polyethylene glycol (PEG), also known as poly(oxyethylene) glycol, is acondensation polymer of ethylene oxide and water having the generalchemical formula HO(CH₂CH₂O)[n]H.

Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates byreplacement of the acetate groups with hydroxyl groups and has theformula (CH₂CHOH)[n].

Most polyvinyl alcohols are soluble in water. PEG, PVA and PVP arecommercially available from chemical suppliers such as the SigmaChemical Company (St. Louis, Mo.).

In certain embodiments the particles may comprisepoly(lactic-co-glycolic acid) (PLGA).

The carrier may be composed of inorganic materials. Inorganic materialsinclude, for instance, magnetic materials, conductive materials, andsemiconductor materials.

In addition to particles the carrier may be composed of any organiccarrier, including biological and living carriers such as cells,viruses, bacteria, as well as any non-living organic carriers, or anycomposition enabling exposure of enzyme substrates to enzymes in disease(including extracellular, membrane-bound, and intracellular enzymes).

In some embodiments, the particles are porous. A porous particle can bea particle having one or more channels that extend from its outersurface into the core of the particle. In some embodiments, the channelmay extend through the particle such that its ends are both located atthe surface of the particle. These channels are typically formed duringsynthesis of the particle by inclusion followed by removal of a channelforming reagent in the particle.

The size of the pores may depend upon the size of the particle. Incertain embodiments, the pores have a diameter of less than 15 microns,less than 10 microns, less than 7.5 microns, less than 5 microns, lessthan 2.5 microns, less than 1 micron, less than 0.5 microns, or lessthan 0.1 microns. The degree of porosity in porous particles may rangefrom greater than 0 to less than 100% of the particle volume. The degreeof porosity may be less than 1%, less than 5%, less than 10%, less than15%, less than 20%, less than 25%, less than 30%, less than 35%, lessthan 40%, less than 45%, or less than 50%. The degree of porosity can bedetermined in a number of ways. For example, the degree of porosity canbe determined based on the synthesis protocol of the carriers (e.g.,based on the volume of the aqueous solution or other channel-formingreagent) or by microscopic inspection of the carriers post-synthesis.

The plurality of particles may be homogeneous for one or more parametersor characteristics. A plurality that is homogeneous for a givenparameter, in some instances, means that particles within the pluralitydeviate from each other no more than about +/−10%, preferably no morethan about +/−5%, and most preferably no more than about +/−1% of agiven quantitative measure of the parameter. As an example, theparticles may be homogeneously porous. This means that the degree ofporosity within the particles of the plurality differs by not more than+/−10% of the average porosity. In other instances, a plurality that ishomogeneous means that all the particles in the plurality were treatedor processed in the same manner, including for example exposure to thesame agent regardless of whether every particle ultimately has all thesame properties. In still other embodiments, a plurality that ishomogeneous means that at least 80%, preferably at least 90%, and morepreferably at least 95% of particles are identical for a givenparameter.

The plurality of particles may be heterogeneous for one or moreparameters or characteristics. A plurality that is heterogeneous for agiven parameter, in some instances, means that particles within theplurality deviate from the average by more than about +/−10%, includingmore than about +/−20%. Heterogeneous particles may differ with respectto a number of parameters including their size or diameter, their shape,their composition, their surface charge, their degradation profile,whether and what type of agent is comprised by the particle, thelocation of such agent (e.g., on the surface or internally), the numberof agents comprised by the particle, etc. The invention contemplatesseparate synthesis of various types of particles which are then combinedin any one of a number of pre-determined ratios prior to contact withthe sample. As an example, in one embodiment, the particles may behomogeneous with respect to shape (e.g., at least 95% are spherical inshape) but may be heterogeneous with respect to size, degradationprofile and/or agent comprised therein.

Particle size, shape and release kinetics can also be controlled byadjusting the particle formation conditions. For example, particleformation conditions can be optimized to produce smaller or largerparticles, or the overall incubation time or incubation temperature canbe increased, resulting in particles which have prolonged releasekinetics.

The particles may also be coated with one or more stabilizingsubstances, which may be particularly useful for long term depoting withparenteral administration or for oral delivery by allowing passage ofthe particles through the stomach or gut without dissolution. Forexample, particles intended for oral delivery may be stabilized with acoating of a substance such as mucin, a secretion containingmucopolysaccharides produced by the goblet cells of the intestine, thesubmaxillary glands, and other mucous glandular cells.

To enhance delivery the particles may be incorporated, for instance,into liposomes, virosomes, cationic lipids or other lipid basedstructures. The term “cationic lipid” refers to lipids which carry a netpositive charge at physiological pH. Such lipids include, but are notlimited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. Additionally,a number of commercial preparations of cationic lipids are available.These include, for example, LIPOFECTIN® (commercially available cationicliposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y.,USA); LIPOFECTAMINE® (commercially available cationic liposomescomprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprising DOGS in ethanol fromPromega Corp., Madison, Wis., USA). A variety of methods are availablefor preparing liposomes e.g., U.S. Pat. Nos. 4,186,183, 4,217,344,4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,4,946,787; and PCT Publication No. WO 91/17424. The particles may alsobe composed in whole or in part of GRAS components. i.e., ingredientsare those that are Generally Regarded As Safe (GRAS) by the US FDA. GRAScomponents useful as particle material include non-degradeable foodbased particles such as cellulose.

The carrier domain can serve several functions. As discussed above, itmay be useful for targeting the product to a specific region, such as atissue. In that instance it could include a targeting agent such as aglycoprotein, an antibody, or a binding protein.

Further, the size of the carrier domain may be adjusted based on theparticular use of the pro-diagnostic reagent. For instance, the carrierdomain may be designed to have a size greater than 5 nm. Particles, forinstance, of greater than 5 nm are not capable of entering the urine,but rather, are cleared through the reticuloendothelial system (RES;liver, spleen, and lymph nodes). By being excluded from the removalthrough the kidneys any uncleaved pro-diagnostic reagent will not bedetected in the urine during the analysis step. Additionally, largerparticles can be useful for maintaining the particle in the blood or ina tumor site where large particles are more easily shuttled through thevasculature. In some embodiments the carrier domain is 500 microns-5 nm,250 microns-5 nm, 100 microns-5 nm, 10 microns-5 nm, 1 micron-5 nm, 100nm-5 nm, 100 nm-10 nm, 50 nm-10 nm or any integer size rangetherebetween. In other instances the carrier domain is smaller than 5 nmin size. In such instance the pro-diagnostic reagent will be clearedinto the urine. However, the presence of free signature molecule canstill be detected for instance using mass spectrometry. In someembodiments the carrier domain is 1-5 nm, 2-5 nm, 3-5 nm, or 4-5 nm.

Optionally the carrier domain may include a biological agent. In oneembodiment a biological agent could be incorporated in the carrierdomain or it may make up the carrier domain. For instance, it may formthe scaffold or platform that the proteolytic domain is attached to.Thus the compositions of the invention can achieve two purposes at thesame time, the diagnostic methods and delivery of a therapeutic agent.In some embodiments the biological agent may be a enzyme inhibitor. Inthat instance the biological agent can inhibit proteolytic activity at alocal site and the signature molecule can be used to test the activityof that particular therapeutic at the site of action. HIV is an exampleof the disease in which active proteases can be monitored. In thisembodiment the composition may include a micro-particle or otherdelivery device carrying a protease inhibitor. The protease susceptiblesite may be sensitive to the HIV proteases such that feedback can beprovided regarding the activity of the particular protease inhibitor.

Biological agents include diagnostic, cosmetic, and therapeutic agents,such as releasable drugs. Thus, any biological agent can be incorporatedwithin the particles, which can locally or systemically deliver ormaintain the incorporated agents following administration or applicationto a subject. Any biocompatible or pharmacologically acceptable materialcan be incorporated into the particles or trapped in the pores of theparticles using technology known to those skilled in the art. Biologicalagents include but are not limited to synthetic inorganic and organiccompounds, proteins and peptides, polysaccharides and other sugars,lipids, and DNA and RNA nucleic acid sequences having therapeutic,prophylactic, cosmetic or diagnostic activities. Nucleic acid sequencesinclude genes, plasmids, vectors, antisense molecules that bind tocomplementary DNA to inhibit transcription, siRNA, shRNA, and ribozymes.

In certain instances, the biological agent is an anti-microbial agent.An anti-microbial agent, as used herein, refers to a naturally-occurringor synthetic compound which is capable of killing or inhibitinginfectious microorganisms. The type of anti-microbial agent usefulaccording to the invention will depend upon the type of microorganismwith which the subject is infected or at risk of becoming infected.Anti-microbial agents include but are not limited to anti-bacterialagents, anti-viral agents, anti-fungal agents and anti-parasitic agents.Phrases such as “anti-infective agent”, “anti-bacterial agent”,“anti-viral agent”, “anti-fungal agent”, “anti-parasitic agent” and“parasiticide” have well-established meanings to those of ordinary skillin the art and are defined in standard medical texts.

Growth factors may also be incorporated into the carrier. As usedherein, the term growth factor refers to any agent that stimulatescellular proliferation and/or differentiation. Growth factors includebut are not limited to fibroblast growth factor (FGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factors (IGF) I and II, TGF-β,TGF-α, bone morphogenetic protein (BMP) (e.g., BMP-2, BMP-3, BMP-4,BMP-6, or BMP-7), hedgehog proteins, growth differentiation factors,hematopoietic colony-stimulating factors (CSF), vascular endotheliumgrowth factor (VEGF), osteoid-inducing factor (OIF), angiogenins,endothelins, hepatocyte growth factor, keratinocyte growth factor,ADMP-1, interleukins (IL) (e.g., IL-3 and IL-6), epithelial growthfactors, dexamethasone, leptin, sortilin, transglutaminase,prostaglandin E, 1,25-dihydroxyvitamin D3, ascorbic acid, pro-collagen,glycerol phosphate, TAK-778, statins, growth hormone, steel factor (SF),activin A (ACT), retinoic acid (RA), epidermal growth factor (EGF),hematopoietic growth factors, peptide growth factors, erythropoietin,tumor necrosis factors (TNF), interferons (IFN), heparin binding growthfactor (HBGF), nerve growth factor (NGF) and muscle morphogenic factor(MMP).

The biological agent may also be an anti-cancer therapy. Anti-cancertherapies include for instance, radiotherapy, chemotherapy, adjuvanttherapy, or any combination of the aforementioned.

The carrier domain may also be configured such that it can be detectedin the body during the analysis. For instance, iron oxide can beincorporated into the particles so that the pro-diagnostic reagent canbe tracked using MRI to provide non-invasive imaging data.

The carrier is linked to the signature producing domain. A signatureproducing domain, as used herein, is the portion of the modularstructure that promotes the enzymatic reaction in the subject, causingthe release of a signature molecule. The signature producing domain iseither an active signature producing agent or an enzyme susceptibledomain linked to a signature molecule. An active signature producingagent is a molecule that causes the formation of a signature molecule bymodifying an endogenous molecule, referred to herein as a biologicalpredictor molecule. For example the active signature producing agent isan enzyme that causes production of a signature molecule when it acts onan endogenous substrate. Enzymes include, for instance, proteases andglycosidases. The biological predictor molecule is the endogenousmolecule acted upon by the active signature producing agent. Biologicalpredictor molecules include for instance, substrates.

The enzyme susceptible site is dependent on enzymes that are active in aspecific disease state. Alternatively, the enzyme specific site may beassociated with enzymes that are ordinarily present but are absent in aparticular disease state. For instance, tumors are associated with aspecific set of enzymes. If the disease state being analyzed is a tumorthen the product is designed with an enzyme susceptible site thatmatches that of the enzyme expressed by the tumor or other diseasedtissue.

An enzyme, as used herein refers to any of numerous proteins produced inliving cells that accelerate or catalyze the metabolic processes of anorganism. Enzymes act on substrates. The substrate binds to the enzymeat a location called the active site just before the reaction catalyzedby the enzyme takes place. Enzymes include but are not limited toproteases, glycosidases, lipases, heparinases, phosphatases.

The enzyme susceptible site may be optimized to provide both highcatalytic activity (or other enzymatic activity) for specified targetenzymes but to also release optimized signature molecules for detection.For the specific detection modality of mass spectrometry, inclusion oftags for rapid affinity purification, specific charges in the sequenceto increase ionization efficiency, mass defects to eliminate endogenousorganic background, and unique mass signatures can improve detection. Inaddition to improving detection sensitivity, by programming the mass ofthese molecules, mass selection techniques can be harnessed to removebackground during detection and focus on expected signature masses.

Patient outcome depends on the phenotype of individual diseases at themolecular level, and this is often reflected in expression of enzymes.The recent explosion of bioinformatics has facilitated exploration ofcomplex patterns of gene expression in human tissues (Fodor, S. A.Massively parallel genomics. Science 277, 393-395 (1997)). Sophisticatedcomputer algorithms have been recently developed capable of moleculardiagnosis of tumors using the immense data sets generated by expressionprofiling (Khan J, Wei J S, Ringner M, Saal L H, Ladanyi M, WestermannF, et al. Classification and diagnostic prediction of cancers using geneexpression profiling and artificial neural networks. Nat Med 2001;7:673-679.). This information can be accessed in order to identifyenzymes and substrates associated with specific diseases. Based on thisinformation the skilled artisan can identify appropriate enzyme orsubstrates to incorporate into the pro-diagnostic reagent. Table 1provides a non-limiting list of enzymes associated with (eitherincreased or decreased with respect to normal) disease and in someinstances, the specific substrate. Table 2 provides a non-limiting listof substrates associated with disease or other conditions. Numerousother enzyme/substrate combinations associated with specific diseases orconditions are known to the skilled artisan and are useful according tothe invention.

TABLE 1 DISEASE ENZYME SUBSTRATE Cancer MMP collagens, gelatin, variousECM proteins Cancer MMP-2 type IV collagen and gelatin Cancer MMP-9 typeIV and V collagens and gelatin Cancer kallikreins kininogens,plasminogen Cancer cathepsins broad spectrum of substrates Cancerplasminogen activator, tPA Plasminogen Cancer ADAM (A Diseintegrin Andvarious extracellular domains Metalloprotease, also MDC, oftransmembrane proteins Adamalysin) Pancreatic carcinoma MMP-7 various,e.g. collagen 18, FasL, HLE, DCN, IGFBP-3, MAG, plasminogen, other MMPsPancreatic Cancer ADAM9, ADAM15 various extracellular domains oftransmembrane proteins Prostate adenocarcinoma Matriptase, a type IIunspecific, cleaves after Lys or transmembrane serine protease Argresidues Prostate cancer Kallikrein 3 kininogens, plasminogen Prostatecancer ADAM15 various extracellular domains of transmembrane proteinsOvarian carcinoma Kallikrein 6 kininogens, plasminogenEpithelial-derived tumors Matriptase, a type II unspecific, cleavesafter Lys or (breast, prostate, transmembrane serine protease Argresidues ovarian, colon, oral) Ovarian Cancer MMP-2, MMP-9,kallikrein-10 type IV and V collagens and (hk-10) gelatin, kininogens,plasminogen Breast, gastric, prostate cancer cathepsins B, L and D broadspectrum of substrates Endometrial cancer cathepsin B unspecificcleavage of a broad spectrum of substrates without clear sequencespecificity esophageal adenocarcinoma cathepsin B unspecific cleavage ofa broad spectrum of substrates without clear sequence specificityInvasive cancers, metastases type II integral serine proteases(dipeptidyl peptidase IV (DPP4/CD26),seprase/fibroblast activationprotein alpha (FAPalpha) and related type II transmembrane prolyl serinepeptidases)) Invasive cancers, metastases Seprase various ECM proteinsviral infections All Retroviruses viral protease precursor GagPol fusionHIV HIV protease (HIV PR, an precursor Gag and GagPol aspartic protease)proteins Hepatitis C NS3 serine protease viral precursor polyproteinDengue Dengue protease auocleavage (NS2B/NS3), NS3/NS4A and NS4B/NS5cleavage West Nile NS2B/NS3pro viral precursor polyprotein bacterialinfections Legionella spp. zinc metalloprotease Me-Arg-Pro-TyrMeninogencephalitis histolytic cysteine protease Streptococcus pyogenesstreptococcal pyrogenic exotoxin extracellular matrix, (Group AStreptococcus) B (SpeB) immunoglobulins, complement componentsChlostridium difficile Cwp84 fibronectin, laminin, vitronectin and otherECM proteins Alzheimer’s disease BACE-1,2 (Alzheimer secretase)β-amyloid precursor protein Stroke and recovery MMP, tPA cardiovasculardisease Angiotensin Converting Enzyme angiotensin I, bradykinin (ACE)Atherosclerosis cathepsin K, L, S broad spectrum of substrates arthritisMMP-1 triple-helical fibrillar collagens rheumatoid arthritis thrombinOsteopontin osteoarthritis thrombin Osteopontinosteoporosis/ostearthritis cathepsin K, S broad spectrum of substratesArthritis, inflammatory joint Aggrecanase (ADAMTS4, aggrecans(proteoglycans) disease ADAMTS11) thrombosis factor Xa (thrombokinase)Prothrombin thrombosis ADAMTS13 von Willebrand factor (vWF) thrombosisplasminogen activator, tPA Plasminogen Stress-induced Renal pressureProstasin epithelial Na channel subunits natriuresis

TABLE 2 DISEASE TARGET SUBSTRATE ENZYME Inflammation Interleukin 1 betaMMP-2, MMP-3, MMP-9, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N,Arg-C Pituitary gland IGFBP-3 MMP-1, MMP-3, MMP-9, dysfunction, abnormalTrypsin, chymotrypsin, pepsin, bone density, growth Lys-C, Glu-C, Asp-N,Arg-C disorders Cancer TGF-beta MMP-9, Trypsin, chymotrypsin, pepsin,Lys-C, Glu-C, Asp-N, Arg-C Cancer, autoimmune TNF MMP-7, Trypsin,chymotrypsin, disease pepsin, Lys-C, Glu-C, Asp-N, Arg-C Cancer,autoimmune FASL MMP-7, Trypsin, chymotrypsin, disease pepsin, Lys-C,Glu-C, Asp-N, Arg-C Wound healing, cardiac HB-EGF MMP-3, Trypsin,chymotrypsin, disease pepsin, Lys-C, Glu-C, Asp-N, Arg-C Pfeiffersyndrome FGFR1 MMP-2, Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C,Asp-N, Arg-C Cancer Decorin MMP-2, MMP-3, MMP-7, Trypsin, chymotrypsin,pepsin, Lys-C, Glu-C, Asp-N, Arg-C Cancer Tumor associatedEndoglycosidases carbohydrate antigens Cancer Sialyl Lewis^(a)O-glycanase Cancer Sialyl Lewis^(X) O-glycanase Cancer/ Rheumatoid VEGFTrypsin, chymotrypsin, pepsin, Arthritis, pulmonary Lys-C, Glu-C, Asp-N,Arg-C hypertension Cancer EGF Trypsin, chymotrypsin, pepsin, Lys-C,Glu-C, Asp-N, Arg-C Cancer IL2 Trypsin, chymotrypsin, pepsin, Lys-C,Glu-C, Asp-N, Arg-C Cancer IL6 Trypsin, chymotrypsin, pepsin,inflammation/angiogenesis Lys-C, Glu-C, Asp-N, Arg-C Cancer IFN-γTrypsin, chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C Cancer TNF-αTrypsin, chymotrypsin, pepsin, inflammation/angiogenesis, Lys-C, Glu-C,Asp-N, Arg-C Rheumatoid Arthritis Cancer, Pulmonary TGF-β Trypsin,chymotrypsin, pepsin, fibrosis, Asthma Lys-C, Glu-C, Asp-N, Arg-CCancer, Pulmonary PDGF Trypsin, chymotrypsin, pepsin, hypertensionLys-C, Glu-C, Asp-N, Arg-C Cancer, pulmonary Fibroblast growth factorTrypsin, chymotrypsin, pepsin, cystadenoma (FGF) Lys-C, Glu-C, Asp-N,Arg-C Cancer Brain-derived neurotrophic Trypsin, chymotrypsin, pepsin,factor (BDNF) Lys-C, Glu-C, Asp-N, Arg-C Cancer Interferon regulatoryTrypsin, chymotrypsin, pepsin, factors (IRF-1, IRF-2) Lys-C, Glu-C,Asp-N, Arg-C Inhibitor of tumor MIF Trypsin, chymotrypsin, pepsin,suppressors Lys-C, Glu-C, Asp-N, Arg-C Lymphomas/carcinomas, GM-CSFTrypsin, chymotrypsin, pepsin, alveolar proteinosis Lys-C, Glu-C, Asp-N,Arg-C Cancer invasion M-CSF Trypsin, chymotrypsin, pepsin, Lys-C, Glu-C,Asp-N, Arg-C Chemical carcinogenesis, IL-12 Trypsin, chymotrypsin,pepsin, multiple schlerosis, Lys-C, Glu-C, Asp-N, Arg-C rheumatoidarthritis, Crohn’s disease Natural Killer T cell IL-15 Trypsin,chymotrypsin, pepsin, leukemias, inflammatory Lys-C, Glu-C, Asp-N, Arg-Cbowel disease, rheumatoid arthritis Cirrhosis Tissue inhibitor of MMPsTrypsin, chymotrypsin, pepsin, (TIMPs) Lys-C, Glu-C, Asp-N, Arg-CCirrhosis Collagen I, III MMP-1, MMP-8, Trypsin, chymotrypsin, pepsin,Lys-C, Glu-C, Asp-N, Arg-C Cirrhosis Collagen IV, V MMP-2, Trypsin,chymotrypsin, pepsin, Lys-C, Glu-C, Asp-N, Arg-C

Several of the enzyme/substrates described above are described in thefollowing publications, all of which are incorporated herein in theirentirety by reference: Parks, W. C. and R. P. Mecham (Eds): Matrixmetalloproteinases. San Diego: Academic Press; 1998; Nagase, H. and J.F. Woessner, Jr. (1999) J. Biol. Chem. 274:21491; Ito, A. et al. (1996)J. Biol. Chem. 271:14657; Schonbeck, U. et al. (1998) J. Immunol. 161:3340; Rajah, R. et al. (1999) Am. J. Cell Mol. Biol. 20:199; Fowlkes, J.L. et al. (1994) Endocrinology 135:2810; Manes, S. et al. (1999) J.Biol. Chem. 274:6935; Mira, E. et al. (1999) Endocrinology 140:1657; Yu,Q. and I. Stamenkovic (2000) Genes Dev. 14:163; Haro, H. et al. (2000)J. Clin. Invest. 105:143; Powell, C. P. et al. (1999) Curr. Biol.9:1441; Suzuki, M. et al. (1997) J. Biol. Chem. 272:31730; Levi, E. etal. (1996) Proc. Natl. Acad. Sci. USA 93:7069; Imai, K. et al. (1997)Biochem. J. 322:809; Smith, M. M. et al. (1995) J. Biol. Chem. 270:6440;and Dranoff, G. (2004) Nat. Rev. Cancer 4: 11-22.

The signature producing domain may be attached directly to the carrier.For instance it may be coated directly on the surface of microparticlesusing known techniques. Alternatively if the carrier is a proteinmaterial it may be directly connected through a peptide bond.Additionally, the signature producing domain may be connected to thecarrier domain through the use of a linker. As used herein “linked” or“linkage” means two entities are bound to one another by anyphysicochemical means. Any linkage known to those of ordinary skill inthe art, covalent or non-covalent, is embraced. Thus, in someembodiments the carrier has a linker attached to an external surface,which can be used to link the signature producing domain. Anothermolecule can also be attached to the linker.

The signature producing domain is preferably a polymer made up of aplurality of chemical units. A “chemical unit” as used herein is abuilding block or monomer which may be linked directly or indirectly toother building blocks or monomers to form a polymer. In some embodimentsthe signature producing domain is a peptide that is susceptible tocleavage by an enzyme or causes cleavage of a substrate associated witha disease or condition. A number of examples of when the proteolyticcleavage site is a peptide are presented in the table above.

The enzyme susceptible domain may also be a polysaccharide. Somepolysaccharide specific degrading enzymes are associated with tumors,angiogenesis and other conditions. A “polysaccharide” is a biopolymercomprised of linked saccharide or sugar units. The polysaccharides usedas proteolytic susceptible domains may be isolated or synthesized denovo. For example, the polysaccharides may be isolated from naturalsources e.g. purified, as by cleavage and gel separation or may besynthesized e.g., by chemical synthesis and incorporated into thepro-diagnostic reagent.

For instance, HSGAG degrading enzymes are enzymes that can be analyzedaccording to the methods of the invention. HSGAG degrading enzymesinclude heparinase-I, heparinase-II, heparinase-III, D-glucuronidase andL-iduronidase. The heparinases cleave at the glycosidic linkage before auronic acid. Heparinase I clips at a glycosidic linkage before a 2-Osulfated iduronic acid. Heparinase-III cleaves at a glycosidic linkagebefore an unsulfated glucuronic acid. Heparinase-II cleaves at bothHep-I and Hep-III cleavable sites. Glucuronidase and iduronidase, astheir name suggests cleave at the glycosidic linkage after a glucuronicacid and iduronic acid respectively. Nitrous acid clips randomly atglycosidic linkages after a N-sulfated hexosamine and converts the sixmembered hexosamine ring to a 5 membered anhydromannitol ring.Appropriate enzyme susceptible domains may be designed based on theknown substrates and cleavage sites of these enzymes.

The pro-diagnostic reagent may also include an implantable microdeliverydevice that houses the modular structure. An implantable microdeliverydevice is any type of device, that is sized for implantation into a bodyand can retain the modular structure. For instance the device may be animplantable capsule that contains the modular structure housed there in.The capsule may have a semi-permeable membrane, such that the modularstructure cannot pass though the membrane, but which is permeable toendogenous molecules such as enzymes and substrates as well as signaturemolecules. Alternatively the implantable microdelivery device may be achip having the modular structure attached thereto. Examples ofimplantable microdelivery devices include but are not limited toimplantable capsules, chips, sustained-release formulations, multi-pulsedrug delivery resorbable polymeric microchip device (Grayson et al.Nature Materials, VOL 2, November 2003, p. 767), and controlled releasemicrochips (Santini et al Nature, vol 397, 1999, p.335). These devicesmay be made from many materials include many of the polymeric materialsdescribed herein. Preferably the implantable devices are biocompatibleand non-toxic.

Modification of the enzyme susceptible domain by an enzyme in vivo,results in the production of a signature molecule. Alternatively, whenthe signature producing domain is an enzyme the enzyme cleaves anendogenous substrate producing a signature molecule from the endogenoussubstrate. The signature molecule is a detectable molecule. It can bepart of the enzyme susceptible domain, e.g. the piece that is releasedor added upon cleavage or it can be a separate entity. The signaturemolecule may be, for instance, a peptide, nucleic acid, small molecule,fluorophore/quencher, carbohydrate, particle, radiolabel, MRI-activecompound, inorganic material, organic material, with encodedcharacteristics to facilitate optimal detection.

The signature molecule may be detected by any known detection methods. Avariety of methods may be used, depending on the nature of the signaturemolecule/label. Labels on signature molecules may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, or signature molecules may be indirectly detected withantibody conjugates, strepavidin-biotin conjugates, mass spectrometry,liquid chromatography-mass spectrometry, PCR analysis, DNA microarray,and fluorescence analysis.

The analysis step may be performed directly on the biological sample orthe signature component may be purified to some degree first. Forinstance, a purification step may involve isolating the signaturemolecule from other components in the biological sample. Purificationsteps include methods such as affinity chromatography. As used herein an“isolated molecule” or “purified molecule” is a signature molecule thatis isolated to some extent from its natural environment. The isolated orpurified molecule need not be 100% pure or even substantially pure priorto analysis.

The methods for analysing signature molecules by identifying thepresence of a signature molecule may be used to provide a qualitativeassessment of the molecule (e.g., whether the signature molecule ispresent or absent) or a quantitative assessment (e.g., the amount ofsignature molecule present to indicate a comparative activity level ofthe enzymes. The quantitative value may be calculated by any means, suchas, by determining the percent relative amount of each fraction presentin the sample. Methods for making these types of calculations are knownin the art.

A signature molecule can be determined using mass spectrometry. The massspectrometry data may be a valuable tool to ascertain information aboutthe signature molecule in the biological sample. After a molecularweight of a signature molecule is identified, it may be compared tomolecular weights of other known signature molecules.

Molecular weight may be determined by several methods including massspectrometry. The use of mass spectrometry for determining the molecularweight of molecules is well known in the art. Liquid chromatography-massspectrometry (LC-MS, or alternatively HPLC-MS) is an analyticalchemistry technique that combines the physical separation capabilitiesof liquid chromatography (or HPLC) with the mass analysis capabilitiesof mass spectrometry. LC-MS is a powerful technique which has very highsensitivity and specificity. It can be used to detect signaturemolecules in a complex mixture. Other types of mass spectrometry knownin the art, such as, matrix-assisted laser desorption ionization massspectrometry (MALDI-MS), electron spray-MS, fast atom bombardment massspectrometry (FAB-MS) and collision-activated dissociation massspectrometry (CAD) can also be used to identify the molecular weight ofthe signature molecule.

Methods for performing mass spectrometry using nucleic acid samples havebeen described. See e.g., U.S. Pat. No. 5,885,775. U.S. Pat. Nos.7,412,332 and 6,597,996 describe methods for detecting polysaccharidemolecules using mass spectrometry. As shown in these patentapplications, one technique for comparing molecular weights is togenerate a mass line and compare the molecular weight of the unknownpolysaccharide to the mass line to determine a subpopulation ofpolysaccharides which have the same molecular weight. A “mass line” isan information database, preferably in the form of a graph or chartwhich stores information for each possible type of polysaccharide havinga unique sequence based on the molecular weight of the polysaccharide.Because mass spectrometry data indicates the mass of a fragment to 1 Daaccuracy, a length may be assigned uniquely to a fragment by looking upa mass on the mass line.

NMR spectroscopy is an analytical tool that allows for the determinationof molecular structure. Utilizing the magnetic properties of somenuclei, the nuclear spins of the nuclei can be oriented randomly with anexternal magnetic field. Oriented nuclei that are subsequentlyirradiated at the correct frequency will absorb energy and transition toa higher energy state. Upon relaxation this energy is emitted anddetected in various NMR systems. This irradiation of the nuclei occur inpulses. In basic one dimensional (1D) NMR the excitation is producedfrom a single pulse and emitted radiation is detected as free inductiondecay (FID). In two dimensional (2D) NMR spectroscopy the nuclei isirradiated with two pulses, and acquisition of the FID occurs at manytime points with a delay between the pulses.

When the signature molecule is a nucleic acid, it can also be analyzedusing PCR and microarrays. PCR methods are well-known in the art. Forinstance, U.S. Pat. No. 5,333,675, issued to Mullis et al. describes anapparatus and method for performing automated PCR. In general,performance of a PCR method results in amplification of a selectedregion of DNA by providing two DNA primers, each of which iscomplementary to a portion of one strand within the selected region ofDNA. The primer is hybridized to a template strand of nucleic acid inthe presence of deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, anddTTP) and a chain extender enzyme, such as DNA polymerase. The primersare hybridized with the separated strands, forming DNA molecules thatare single stranded except for the region hybridized with the primer,where they are double stranded. The double stranded regions are extendedby the action of the chain extender enzyme (e.g. DNA polymerase) to forman extended double stranded molecule between the original two primers.The double stranded DNA molecules are separated to produce singlestrands which can then be re-hybridized with the primers. The process isrepeated for a number of cycles to generate a series of DNA strandshaving the same nucleotide sequence between and including the primers.

Chain extender enzymes are well known in the art and include, forexample, E. coli DNA polymerase I, klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, T7 DNA polymerase, recombinant modifiedT7 DNA polymerase, reverse transcriptase, and other enzymes. Heat stableenzymes are particularly preferred as they are useful in automatedthermal cycle equipment. Heat stable polymerases include, for example,DNA polymerases isolated from Bacillus stearothermophilus (Bio-Rad),Thermus thermophilous (finzyme, ATCC number 27634), Thermus species(ATCC number 31674), Thermus aquaticus strain TV11518 (ATCC number25105), Sulfolobus acidocaldarius, described by Bukhrashuili et al.,Biochem. Biophys. Acta., 1008:102-07 (1909), Therms filiformus (ATCCnumber 43280), Taq DNA polymerase, commercially available fromPerkin-Elmer-Cetus (Norwalk, Conn.), Promega (Madison, Wis.) andStratagene (La Jolla, Calif.), and AmpliTaq™ DNA polymerase, arecombinant thermus equitus Taq DNA polymerase, available fromPerkin-Elmer-Cetus and described in U.S. Pat. No. 4,889,818.

Preferably, the PCR-based methods performed according to the inventionare automated and performed using thermal cyclers. Many types of thermalcyclers are well-known in the art. For instance, M.J. Research(Watertown, Mass.) provides a thermal cycler having a peltier heat pumpto provide precise uniform temperature control in the thermal cyclers;DeltaCycler thermal cyclers from Ericomp (San Diego, Calif.) also arepeltier-based and include automatic ramping control, time/temperatureextension programming and a choice of tube or microplate configurations.The RoboCycler™ by Stratagene (La Jolla, Calif.) incorporates roboticsto produce rapid temperature transitions during cycling and well-to-welluniformity between samples; and a particularly preferred cycler, is thePerkin-Elmer Applied Biosystems (Foster City, Calif.) ABI Prism™ 877Integrated Thermal cycler, which is operated through a programmableinterface that automates liquid handling and thermocycling processes forfluorescent DNA sequencing and PCR reactions.

The presence or absence of enzymes in the subject may also be determinedusing hybridization techniques. Standard hybridization techniques ofmicroarray technology are utilized to assess the presence of nucleicacids in the biological sample. Microarray technology, which is alsoknown by other names including: DNA chip technology, gene chiptechnology, and solid-phase nucleic acid array technology, is well knownto those of ordinary skill in the art and is based on, but not limitedto, obtaining an array of identified nucleic acid probes on a fixedsubstrate, labeling target molecules with reporter molecules (e.g.,radioactive, chemiluminescent, or fluorescent tags such as fluorescein,Cye3-dUTP, or Cye5-dUTP), hybridizing target nucleic acids to theprobes, and evaluating target-probe hybridization. A probe with anucleic acid sequence that perfectly matches the target sequence will,in general, result in detection of a stronger reporter-molecule signalthan will probes with less perfect matches. Many components andtechniques utilized in nucleic acid microarray technology are presentedin The Chipping Forecast, Nature Genetics, Vol. 21, January 1999, theentire contents of which is incorporated by reference herein.

According to the present invention, microarray substrates may includebut are not limited to glass, silica, aluminosilicates, borosilicates,metal oxides such as alumina and nickel oxide, various clays,nitrocellulose, or nylon. In all embodiments a glass substrate ispreferred. An “array” as used herein is a set of molecules arranged in aspecific order with respect to a surface. Preferably the array iscomposed of polynucleotides attached to the surface. Oligonucleotidearrays can be used to screen nucleic acid samples for a target nucleicacid, which can be labeled with a detectable marker. A fluorescentsignal resulting from hybridization between a target nucleic acid and asubstrate-bound oligonucleotide provides information relating to theidentity of the target nucleic acid by reference to the location of theoligonucleotide in the array on the substrate. Such a hybridizationassay can generate thousands of signals which exhibit different signalstrengths. These signals correspond to particular oligonucleotides ofthe array. Different signal strengths will arise based on the amount oflabeled target nucleic acid hybridized with an oligonucleotide of thearray.

Conditions for optimal hybridization are known. The hybridizationconditions in general are those used commonly in the art, such as thosedescribed in Sambrook et al., “Molecular Cloning: A Laboratory Manual”,(1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, “Guide toMolecular Cloning Techniques”, Methods in Enzymology, (1987), Volume152, Academic Press, Inc., San Diego, Calif.; and Young and Davis,(1983), PNAS (USA) 80:1194. In general, incubation temperatures forhybridization of nucleic acids range from about 20° C. to 75° C. Forprobes 17 nucleotides residues and longer, a preferred temperature rangefor hybridization is from about 50° C. to 54° C. The hybridizationtemperature for longer probes is preferably from about 55° C. to 65° C.and for shorter probes is less than 52° C. Rehybridization may beperformed in a variety of time frames. Preferably, hybridization of SNPand RCGs performed for at least 30 minutes.

The signature molecule may be labeled. For example, a label may be addeddirectly to a nucleic acid when the isolated signature molecule issubjected to PCR. For instance, a PCR reaction performed using labeledprimers or labeled nucleotides will produce a labeled product. Labelednucleotides (e.g., fluorescein-labeled CTP) are commercially available.Methods for attaching labels to nucleic acids are well known to those ofordinary skill in the art and, in addition to the PCR method, include,for example, nick translation and end-labeling.

Labels suitable for use in the methods of the present invention includeany type of label detectable by standard means, including spectroscopic,photochemical, biochemical, electrical, optical, or chemical methods.Preferred types of labels include fluorescent labels such asfluorescein. A fluorescent label is a compound comprising at least onefluorophore. Commercially available fluorescent labels include, forexample, fluorescein phosphoramidides such as fluoreprime (Pharmacia,Piscataway, N.J.), fluoredite (Millipore, Bedford, Mass.), FAM (ABI,Foster City, Calif.), rhodamine, polymethadine dye derivative,phosphores, Texas red, green fluorescent protein, CY3, and CY5.Polynucleotides can be labeled with one or more spectrally distinctfluorescent labels. “Spectrally distinct” fluorescent labels are labelswhich can be distinguished from one another based on one or more oftheir characteristic absorption spectra, emission spectra, fluorescentlifetimes, or the like. Spectrally distinct fluorescent labels have theadvantage that they may be used in combination (“multiplexed”).Radionuclides such as ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P are also useful labelsaccording to the methods of the invention. A plurality of radioactivelydistinguishable radionuclides can be used. Such radionuclides can bedistinguished, for example, based on the type of radiation (e.g. α, β,or δ radiation) emitted by the radionuclides. The ³²P signal can bedetected using a phosphoimager, which currently has a resolution ofapproximately 50 microns. Other known techniques, such aschemiluminescence or colormetric (enzymatic color reaction), can also beused.

Quencher compositions in which a “donor” fluorophore is joined to an“acceptor” chromophore by a short bridge that is the binding site forthe enzyme may also be used. The signal of the donor fluorophore isquenched by the acceptor chromophore through a process believed toinvolve resonance energy transfer (RET). Cleavage of the peptide resultsin separation of the chromophore and fluorophore, removal of the quench,and generation of a subsequent signal measured from the donorfluorophore.

Once the data is obtained, e.g. as a two-dimensional image, a computercan be used to transform the data into a displayed image which varies incolor depending on the intensity of light emission at a particularlocation. Any type of commercial software which can perform this type ofdata analysis can be used. In general, the data analysis involves thesteps of determining the intensity of the fluorescence emitted as afunction of the position on the substrate, removing the outliers, andcalculating the relative binding affinity. One or more of the presence,absence, and intensity of signal corresponding to a label is used toassess the presence or absence of an signature molecule. The presenceand absence of one or more signature molecules can be used to determinethe disease status of an individual based on the presence or absence ofan enzyme.

The data may also be observed and analyzed manually. For instance, thepresence or absence of a fluorescent label may be observed in order toprovide the diagnostic or prognostic information from the data.

The disease or condition assessed acccording to the methods of theinvention is any disease or condition that is associated with an enzyme.For instance, cancer, cardiovascular disease, arthritis, viral,bacterial, parasitic or fungal infection, Alzheimer's disease emphysema,thrombosis, hemophilia, stroke, organ disfunction, any inflammatorycondition, vascular disease, parenchymal disease, or apharmacologically-induced state are all known to be associated withenzymes. A pharmacologically induced state is a condition in whichenzyme inhibitors and other agents directly or indirectly affect enzymeactivities. Thus each of the these can be assessed or monitored orstudied according to methods of the invention.

It is useful to be able to differentiate non-metastatic primary tumorsfrom metastatic tumors, because metastasis is a major cause of treatmentfailure in cancer patients. If metastasis can be detected early, it canbe treated aggressively in order to slow the progression of the disease.Metastasis is a complex process involving detachment of cells from aprimary tumor, movement of the cells through the circulation, andeventual colonization of tumor cells at local or distant tissue sites.Additionally, it is desirable to be able to detect a predisposition fordevelopment of a particular cancer such that monitoring and earlytreatment may be initiated. For instance, an extensive cytogeneticanalysis of hematologic malignancies such as lymphomas and leukemiashave been described, see e.g., Solomon et al., Science 254, 1153-1160,1991. Early detection or monitoring using the non-invasive methods ofthe invention may be useful.

Solid tumors progress from tumorigenesis through a metastatic stage andinto a stage at which several different active proteases can beinvolved. Some protease are believed to alter the tumor such that it canprogress to the next stage, i.e., by conferring proliferativeadvantages, the ability to develop drug resistance or enhancedangiogenesis, proteolysis, or metastatic capacity.

Alzheimer's disease causes progressive dementia with consequentformation of amyloid plaques, neurofibrillary tangles, gliosis andneuronal loss. The disease occurs in both genetic and sporadic formswhose clinical course and pathological features are quite similar. Threegenes have been discovered to date which, when mutated, cause anautosomal dominant form of Alzheimer's disease. These encode the amyloidprotein precursor (APP) and two related proteins, presenilin-1 (PS1) andpresenilin-2 (PS2). Mutations in any of the three proteins have beenobserved to enhance proteolytic processing of APP via an intracellularpathway that produces amyloid beta peptide (Aβ peptide), a 40-42 aminoacid long peptide that is the primary component of amyloid plaque inAlzheimer's disease. Pathological processing of APP at the β- andγ-secretase sites, which are located N-terminal and C-terminal to theα-secretase site, respectively, produces a very different result thanprocessing at the α site. Sequential processing at the β- andγ-secretase sites releases the Aβ peptide, a peptide possibly veryimportant in Alzheimer's disease pathogenesis. The β secretase enzyme,termed Aspartyl Protease 2 (Asp2) is thought to mediate this processing.The presence of Asp2 activity is important for the diagnosis andprognosis of Alzheimer's disease. This enzyme and it's substrate canalso be used in the methods of the invention to monitor the ability of atherapeutic to function in slowing the progression of Alzheimer'sdisease.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat, or rodent. In all embodiments human subjectsare preferred. In aspects of the invention pertaining to cancerdiagnosis in general the subject preferably is a human suspected ofhaving cancer, or a human having been previously diagnosed as havingcancer. Methods for identifying subjects suspected of having cancer mayinclude physical examination, subject's family medical history,subject's medical history, biopsy, or a number of imaging technologiessuch as ultrasonography, computed tomography, magnetic resonanceimaging, magnetic resonance spectroscopy, or positron emissiontomography.

As used herein, a biological sample is a tissue sample. The biologicalsample may be examined in the body, for instance, by detecting a labelat the site of the tissue, i.e. urine. Alternatively the biologicalsample may be collected from the subject and examined in vitro.Biological samples include but are not limited to urine, blood, saliva,or mucous secretion. In preferred embodiments the tissue sample isobtained non-invasively, such as the urine.

A “plurality” of elements, as used throughout the application refers to2 or more of the elements.

The pro-diagnostic reagents of the invention are administered to thesubject in an effective amount for detecting enzyme activity. An“effective amount”, for instance, is an amount necessary or sufficientto cause release of a detectable level of signature molecule in thepresence of an enzyme. In some instances when a therapeutic isadministered in the pro-diagnostic reagent, the effective amount is thatamount necessary to realize a desired biologic effect. An “effectiveamount for treating Alzheimer's disease”, for instance, could be thatamount necessary to (i) prevent further memory loss and/or (ii)arresting or slowing memory loss with respect to memory loss in theabsence of the therapy. According to some aspects of the invention, aneffective amount is that amount of a compound of the invention alone orin combination with another medicament, which when combined orco-administered or administered alone, results in a therapeutic responseto the disease, either in the prevention or the treatment of thedisease. The biological effect may be the amelioration and or absoluteelimination of symptoms resulting from the disease. In anotherembodiment, the biological effect is the complete abrogation of thedisease, as evidenced for example, by the absence of a symptom of thedisease.

The effective amount of a compound of the invention described herein mayvary depending upon the specific compound used, the mode of delivery ofthe compound, and whether it is used alone or in combination. Theeffective amount for any particular application can also vary dependingon such factors as the disease being assessed or treated, the particularcompound being administered, the size of the subject, or the severity ofthe disease or condition as well as the detection method. One ofordinary skill in the art can empirically determine the effective amountof a particular molecule of the invention without necessitating undueexperimentation. Combined with the teachings provided herein, bychoosing among the various active compounds and weighing factors such aspotency, relative bioavailability, patient body weight, severity ofadverse side-effects and preferred mode of administration, an effectiveregimen can be planned.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. Moreover, for animal (e.g., human) administration, itwill be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences(1990), incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated. Theagent may comprise different types of carriers depending on whether itis to be administered in solid, liquid or aerosol form, and whether itneed to be sterile for such routes of administration as injection.

Preferably the material is injected into the body but could also beadministered by other routes. For instance, the compounds of the presentinvention can be administered intravenously, intradermally,intraarterially, intralesionally, intratumorally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in creams, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences (1990), incorporated herein byreference).

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein.

The agent may be formulated into a composition in a free base, neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

According to the methods of the invention, the compound may beadministered in a pharmaceutical composition. In general, apharmaceutical composition comprises the compound of the invention and apharmaceutically-acceptable carrier. Pharmaceutically-acceptablecarriers for peptides, monoclonal antibodies, and antibody fragments arewell-known to those of ordinary skill in the art. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Exemplary pharmaceutically acceptable carriersfor peptides in particular are described in U.S. Pat. No. 5,211,657.Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The compounds of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

The compounds of the invention may be administered directly to a tissue.Direct tissue administration may be achieved by direct injection. Thecompounds may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the compounds may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. Techniques forpreparing aerosol delivery systems are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the active agent(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporatedby reference). Those of skill in the art can readily determine thevarious parameters and conditions for producing aerosols without resortto undue experimentation.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

The data generated according to the invention may optionally beconverted into a bar code or other human- or machine-readable form. Forexample, each line of a bar code may indicate the presence or absence ofa specific enzyme or groups of specific enzymes for a particularsubject. The bar code data can be compared with a database ofinformation on other subjects or information on the disease to aid inthe diagnosis, prognosis or other analysis of the test subject.

In one embodiment of the invention, the data generated herein is used toselect clinical treatment paradigms for cancers. Treatment options, asdescribed herein, may include but are not limited to: radiotherapy,chemotherapy, adjuvant therapy, or any combination of the aforementionedmethods. Aspects of treatment that may vary include, but are not limitedto: dosages, timing of administration, or duration or therapy; and mayor may not be combined with other treatments, which may also vary indosage, timing, or duration. Another treatment for cancer is surgery,which can be utilized either alone or in combination with any of theaforementioned treatment methods. One of ordinary skill in the medicalarts may determine an appropriate treatment paradigm based on evaluationof data generated by the methods described herein.

In certain embodiments, software for calculating and processing the dataas described herein can be provided on a computer connected by data linkto a data generating device, such as a mass spectrometer, microarrayreader or PCR machine. Any standard data link can be used, includingserial or parallel cables, radio frequency or infrared telemetry links,LAN connections, WAN connections, etc. Alternatively, data can betransferred by computer-readable medium (e.g., magnetic or opticalmedium) and read by the software. The data also can be entered directlyby the user via user interface, such as a keyboard, monitor, mouse,graphical user interface such as touch screen, etc. The computer may becontained within the data generating device, providing an integratedsystem for generating raw data, calculating ratios, and displaying suchratios. One or more computers also may be linked to one or more datagenerating devices and one or more display devices, such as in a localarea network or wide area network.

In one embodiment of the invention, a visual display is used to displaythe data for the classification, diagnosis, prediction of prognosisand/or therapeutic monitoring. The visual display can be a graphicaluser interface, such as a monitor, or a printer.

The data can be processed individually or by a computer. For instance, acomputer-implemented method for generating a data structure, tangiblyembodied in a computer-readable medium, representing a quantitativevalue of a set of signature molecules may be performed according to theinvention. The quantitative values may be compared with a referencedatabase. Alternatively a qualitative pattern may be compared with areference database.

A computer system that may implement the above as a computer programtypically may include a main unit connected to both an output devicewhich displays information to a user and an input device which receivesinput from a user. The main unit generally includes a processorconnected to a memory system via an interconnection mechanism. The inputdevice and output device also may be connected to the processor andmemory system via the interconnection mechanism.

One or more output devices may be connected to the computer system.Example output devices include a cathode ray tube (CRT) display, liquidcrystal displays (LCD), printers, communication devices such as a modem,and audio output. One or more input devices also may be connected to thecomputer system. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication device, and data inputdevices such as sensors. The subject matter disclosed herein is notlimited to the particular input or output devices used in combinationwith the computer system or to those described herein.

The computer system may be a general purpose computer system which isprogrammable using a computer programming language, such as C++, Java,or other language, such as a scripting language or assembly language.The computer system also may include specially-programmed, specialpurpose hardware such as, for example, an Application-SpecificIntegrated Circuit (ASIC). In a general purpose computer system, theprocessor typically is a commercially-available processor, of which theseries x86, Celeron, and Pentium processors, available from Intel, andsimilar devices from AMD and Cyrix, the 680X0 series microprocessorsavailable from Motorola, the PowerPC microprocessor from IBM and theAlpha-series processors from Digital Equipment Corporation, areexamples. Many other processors are available. Such a microprocessorexecutes a program called an operating system, of which Windows NT,Linux, UNIX, DOS, VMS and OS8 are examples, which controls the executionof other computer programs and provides scheduling, debugging,input/output control, accounting, compilation, storage assignment, datamanagement and memory management, and communication control and relatedservices. The processor and operating system define a computer platformfor which application programs in high-level programming languages maybe written.

A memory system typically includes a computer readable and writeablenonvolatile recording medium, of which a magnetic disk, a flash memoryand tape are examples. The disk may be removable, such as a “floppydisk,” or permanent, known as a hard drive. A disk has a number oftracks in which signals are stored, typically in binary form, i.e., aform interpreted as a sequence of one and zeros. Such signals may definean application program to be executed by the microprocessor, orinformation stored on the disk to be processed by the applicationprogram. Typically, in operation, the processor causes data to be readfrom the nonvolatile recording medium into an integrated circuit memoryelement, which is typically a volatile, random access memory such as adynamic random access memory (DRAM) or static memory (SRAM). Theintegrated circuit memory element typically allows for faster access tothe information by the processor than does the disk. The processorgenerally manipulates the data within the integrated circuit memory andthen copies the data to the disk after processing is completed. Avariety of mechanisms are known for managing data movement between thedisk and the integrated circuit memory element, and the subject matterdisclosed herein is not limited to such mechanisms. Further, the subjectmatter disclosed herein is not limited to a particular memory system.

The subject matter disclosed herein is not limited to a particularcomputer platform, particular processor, or particular high-levelprogramming language. Additionally, the computer system may be amultiprocessor computer system or may include multiple computersconnected over a computer network. It should be understood that eachmodule may be separate modules of a computer program, or may be separatecomputer programs. Such modules may be operable on separate computers.Data may be stored in a memory system or transmitted between computersystems. The subject matter disclosed herein is not limited to anyparticular implementation using software or hardware or firmware, or anycombination thereof. The various elements of the system, eitherindividually or in combination, may be implemented as a computer programproduct tangibly embodied in a machine-readable storage device forexecution by a computer processor. Various steps of the process may beperformed by a computer processor executing a program tangibly embodiedon a computer-readable medium to perform functions by operating on inputand generating output. Computer programming languages suitable forimplementing such a system include procedural programming languages,object-oriented programming languages, and combinations of the two.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents active in vivo.Generally, the screening methods involve assaying for compounds thatbeneficially alter enzyme activity in vivo. Such methods according tothe invention are adaptable to automated, high-throughput screening ofcompounds.

The methods may be used in any subject. For instance animal models ofdisease may be used to screen multiple putative therapeutic agents inorder to assess the activity level of the putative therapeutic agents onparticular enzymes associated with disease. For instance, a library ofpro-diagnostic reagents having different putative therapeutic agentsassociated with the carrier can be administered to the animal model. Ifeach therapeutic agent is associated with a unique signature molecule,then the activity of the putative therapeutic agent could be assessed byanalyzing the level of signature molecule in the urine as describedherein.

Additionally, the methods may be used for the advancement ofpersonalized medicine. For instance, a set of pro-diagnostic reagentshaving multiple therapeutic agents, each therapeutic agent associatedwith a discreet signature molecule could be administered to a subjecthaving a disease to assess which therapeutic agent is most effective inthat individual subject. Based on the data, an appropriate therapeuticstrategy could be designed. An example of this may be seen in HIV.Protease inhibitors are used therapeutically to inhibit the activity ofcritical proteases associated with HIV survival and activity. A set ofpro-diagnostic reagents could be generated having different enzymeinhibitors as the carrier or part of the carrier. Each enzyme inhibitoris associated with a particular signature molecule, such that theactivity of the particular inhibitor on the enzyme can be assessed bymonitoring the level of signature in the urine. For instance aparticularly active inhibitor would cause a reduced level of signaturemolecule being transported to the urine.

Typically, a known active therapeutic agent may serves as a negativecontrol, i.e., the known therapeutic agent is incorporated into apro-diagnostic reagent. Putative therapeutic agents, also referred toherein as candidate agents encompass numerous chemical classes, althoughtypically they are organic compounds. Preferably, the candidatepharmacological agents are small organic compounds, i.e., those having amolecular weight of more than 50 yet less than about 2500, preferablyless than about 1000 and, more preferably, less than about 500.Candidate agents comprise functional chemical groups necessary forstructural interactions with polypeptides and/or nucleic acids, andtypically include at least an amine, carbonyl, hydroxyl, or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is anucleic acid, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acids as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

The invention also includes kits having a container housing apro-diagnostic reagent, wherein the pro-diagnostic reagent comprises acarrier domain linked to a signature producing domain. The kit may alsoinclude a second container housing an analytical reagent.

The kits, also referred to as articles, include pharmaceutical ordiagnostic grade compounds of the invention in one or more containers.The article may include instructions or labels promoting or describingthe use of the compounds of the invention. For instance, the kit mayinclude instructions for administering the pro-diagnostic reagent to asubject and for analyzing the signature molecule of the pro-diagnosticreagent in a biological sample of the subject.

As used herein, “promoted” includes all methods of doing businessincluding methods of education, hospital and other clinical instruction,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with compositions ofthe invention.

“Instructions” can define a component of promotion, and typicallyinvolve written instructions on or associated with packaging ofcompositions of the invention. Instructions also can include any oral orelectronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto pharmaceutical or diagnostic or research kits to facilitate theiruse in therapeutic, diagnostic or research applications. A kit mayinclude one or more containers housing the components of the inventionand instructions for use. Specifically, such kits may include one ormore agents described herein, along with instructions describing theintended therapeutic application and the proper administration of theseagents. In certain embodiments agents in a kit may be in apharmaceutical formulation and dosage suitable for a particularapplication and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are sued, the liquidform may be concentrated or ready to use. The solvent will depend on thecompound and the mode of use or administration. Suitable solvents fordrug compositions are well known and are available in the literature.The solvent will depend on the compound and the mode of use oradministration.

The kits, in one set of embodiments, may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the containers may comprise a positive control for anassay. Additionally, the kit may include containers for othercomponents, for example, buffers useful in the assay.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In thecase of dosage forms suitable for parenteral administration the activeingredient is sterile and suitable for administration as a particulatefree solution. In other words, the invention encompasses both parenteralsolutions and lyophilized powders, each being sterile, and the latterbeing suitable for reconstitution prior to injection. Alternatively, theunit dosage form may be a solid suitable for oral, transdermal, topicalor mucosal delivery.

In a preferred embodiment, the unit dosage form is suitable forintravenous, intramuscular or subcutaneous delivery. Thus, the inventionencompasses solutions, preferably sterile, suitable for each deliveryroute.

In another preferred embodiment, compositions of the invention arestored in containers with biocompatible detergents, including but notlimited to, lecithin, taurocholic acid, and cholesterol; or with otherproteins, including but not limited to, gamma globulins and serumalbumins.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures.

More specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material. The invention further providesan article of manufacture comprising a needle or syringe, preferablypackaged in sterile form, for injection of the formulation, and/or apackaged alcohol pad.

EXAMPLES Example 1 Methods

Syntheses of Nanoparticle Pro-Diagnostic Reagents

40 nm dextran-coated iron oxide nanoparticles (NP; 115,000 g/mole periron core) were dissolved in 1× phosphate buffered saline (PBS; 137 mMNaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) at a concentration of 2mg/mL. Vivotag-750 fluorophore was labeled on the NPs such that each NPhas around 2 VT-750 fluorophores. The linker N-succinimidyl iodoacetate(SIA) was dissolved in DMSO at 20 mg/mL. The two solutions were mixed toobtain a 1-to-7 mass ratio between iron oxide NPs and SIA for 2 hr atroom temperature with shaking. Size exclusion chromatography (columndiameter×height=1 cm×30 cm; media: Sephadex G-50-coarse) was used toseparate out the excess SIA and to exchange NPs into borate buffer (50mM sodium borate, 5 mM EDTA, pH 8.3).

Each of the 43 fluorescein-labeled peptides; MIT Biopolymers Laboratory)was dissolved in DMSO at 25 mg/mL. Thiol polyethylene glycol (SH-PEG;MW=20k) was dissolved in borate buffer at 1 mg/mL. Each of the 43peptides, SH-PEGs, and the activated NPs were left to for >12 hr atmolar ratio of 1:20:95=NP:SH-PEG:peptide, making 43 differentpeptide-PEG-NPs. Additional borate buffer was added to the reactants tobring the DMSO to <10% of the total reaction volume.

After the linkers on the NP surface had reacted with 20k SH-PEGs andfluorophore-peptides, the five final-product solutions were filtered oncentrifugal filter columns (Amicon, Millipore; MW=100k) at 4,200 rcf toremove the un-conjugated SH-PEGs and peptides to <0.1% of the originalconjugated quantity. 1× HEPES salt buffer (100 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 150 mM NaCl, pH 7.5)was used to replace the borate buffer and DMSO during centrifugationuntil <0.1% volume of borate and <0.02% volume of the DMSO were left inthe samples.

The 43 newly made peptide-PEG-NPs (pP-NPs) were analyzed with aspectrometer to assess the number of peptides bound to eachnanoparticle. Nanoparticle spectra were normalized to particles that hadnot been reacted with peptides to allow quantification of the attachedfluorophore-peptide absorbance (fluorescein peak absorbance=495 nm;extinction coefficient=72,000×10⁶ cm⁻¹ M⁻¹). NP concentration wasassessed by recording its absorbance at 400 nm with an extinctioncoefficient of 2.07×10⁶ cm⁻¹ M⁻¹, Comparison of these concentrationsallowed quantitation of the average peptide-to-NP ratios. All sampleswere normalized to 5 μM based on peptide concentration and stored in 4°C.

In Vitro Screen of Extracellular Protease Activations on NanoparticlePro-Diagnostic Reagents

The 43 normalized (based on 5 μM peptide concentrations) pP-NPs werealiquoted on a clear half-96-well plate. From the aliquoted “stock”plate, 20 μL of each sample was taken out and put into a black half-area96-well plate. MMP-2 was dissolved in 1× HEPES salt buffer (with 5 mMCaCl₂) at 64 nM, and the solution was heated at 37° C. for 15 minutes toactivate the enzyme. 20 μL of the MMP-2 solution was put into each ofall 43 wells (which already contained the pP-NP samples) on the blackhalf-96-well plate such that each well had 32 nM MMP-2 and 2.5 μMpeptide concentrations. The plate was read immediately and every 1-minfollowing for 90 min by using a microplate fluorimeter (MolecularDevices Corporation; Gemini EM; excitation: 485 nm, emission: 538 nm,cutoff: 530 nm) to sense the cleaved fluorophore signals. The proteaseactivities for particular peptide substrate were measured by thefluorophore intensity levels over time. The basic physical concept isthat when the peptide-fluorophore was attached to the NP iron core, thefluorescence of the fluorophore is quenched by the neighboringabsorption iron cores. However, when the peptide were cleaved (nowde-quench from iron core), an increase in fluorescence can be detectedas it diffuses away from the particle core. Same steps were repeated forMMP-7, MMP-8, MMP-9, and MMP-14.

Other proteases (Thrombin, factor Xa, tissue factor, and Cathepsin B)dissolved in 1× HEPES (but no CaCl₂) were used to test the enzymeactivities at a final concentration of 32 nM. 20 μL Dulbecco's ModifiedEagle Medium (DMEM, GibcoBRL, Rockville, Md.) with 10% fetal bovineserum was put into each well of the prepared black half-96-well plate aswell. The DMEM with 10% serum served as the control for the experiment.The initial slope of time over fluorophore intensity level(V₀=milli-unit per minute) was plotted for each trials from eachsamples. There were total of 43 samples and 10 proteases (MMP-2, MMP-7,MMP-8, MMP-9, MMP-14, thrombin, factor Xa, tissue factor, Cathepsin B,and DMEM with 10% serum). The data were then collected and analyzed, andthe around 10 most effective samples were selected for future in vivoexperiment.

HT-1080/MDA-MB-435 Cancer Cell Culture and Implantation

HT-1080 human fibrosarcoma cells (American Type Culture Collection;ATCC) were cultured in tissue culture flasks (150 cm²) by using cellmedia solution consist of DMEM (89%), fetal bovine serum (10%;Invitrogen), and penicillin/streptomycin (1%). The cells were grown overseveral generation (n=4-5) and were then concentrated to around 2×10⁶million cells/mL in a serum-free solution (66% DMEM, 33% matrix gel, and1% penicillin/streptomycin). 200 μL of cell solution were injectedsubcutaneously into each of the nude mice (Nu/Nu; strain code: 088;Charles River Laboratories, Inc., Wilmington, MA). For each mouse, 100μL of the cell solution were injected into each of the nude mouse on theregions of upper right/left thigh (thus, 200 μL total per mouse) belowthe skin but not into the tissues. The cells would gradually settled andstarted growing into a sizable tumor (diameter=1 cm) in around 14 days.MDA-MB-435 was processed in the exact same way as HT-1080 with oneexception: the injection solution contained 50% 2×10⁶ million cells/mLand 50% DMEM.

Circulation Assessment of Nanoparticle Pro-Diagnostic Reagents In Vivo

Thrombin-specific pP-NPs samples (2 n-mol based on peptideconcentration) were injected into each of four 1-month old adult femalewhite mice (Swiss Webster; strain code: 024; Charles River Laboratories,Inc., Wilmington, Mass.). The reagents (also referred to herein asNP-chaperones) were allowed to circulate in the bloodstream for 5 min(the endpoint of which was defined as 0 hr). 15 μL of blood samples weretaken retro-orbitally with heparinized capillary tubes at the followingtime points: 0 hr, 1 hr, 3 hr, 6 hr, 8 hr, and 12 hr. 10 μL of the bloodsample was mixed gently with 40 μL 1× PBS containing 10 mM EDTA tochelate blood calcium and prevent coagulation; the mixture was vortexedand centrifuged to pellet the red blood cells which were then discarded.30 μL of the supernatant (blood plasma) was put into a well on a24-by-36-microarray plate.

The intensity of the NP-linked Vivotag-750 fluorescence in the bloodplasma sample was detected with the Odyssey imaging system (Westburg,Leusden, Netherlands). All fluorescence intensity levels were normalizedto the 0 hr time point (defined as 100%). Bi-exponential lines of bestfit were drawn for all samples to calculate the half-life points.

In Vivo Assessment of Tumor and Clotting-Factor Protease Activity

Out of the nine selected peptide-PEG-NP (pP-NPs), the first sevenpeptide were targeted for tumor monitoring while last two peptide werechosen for internal, vascular injury assessment. These nine pP-NPs werere-synthesized in larger quantity (at least 100 n-moles based on peptideconcentrations). Each of the nine pP-NPs (around 5 n-moles) was reactedwith VT-750 flourophore (at five-fold molar excess to the peptideconcentrations on the NPs) for 2 hr, and the excess un-reactive VT-750were filtered out to <0.01% by spinning down on 100k filter column. TheVT-750 was now covalently connected to the end of the peptides onpP-NPs, and we abbreviate these products as 750-pP-NPs.

The two thrombin-specific-cleavage 750-pP-NP chaperones were each takenout based on 1-nmol VT-750 concentrations, combined together, andre-suspended to 200 uL of 1× PBS. After fed on non-flourophore dietfor >1 wk, 9 nude mice were each injected intravenously with the 200 uLof the 750-pP-NPs; immediately, 4 of the 9 mice were slightly injured onboth thigh muscles while 1 of the 9 was injured on only one thighmuscles. All mice were imaged for the bio-distribution of NP chaperonesin vivo (Odyssey imaging systems; Westburg, Leusden, Netherlands) bytracing VT-750 for 2-hr period with 10-min intervals. Thebio-distributions of 750-pP-NPs over time in each mouse were quantified(ImageJ; NIH), and the organs of interest included bladder, kidney,liver, and spleen. The seven MMP-specific cleavage 750-pP-NP chaperoneswere each taken out based on 0.3-nmol VT-750 concentrations, combinedtogether, and re-suspended to 200 uL of 1× PBS. After fed onnon-flourophore diet for >1 wk, 9 nude mice (3 with HT-1080 tumors, 3with MDA-MB-435 tumors, and 3 with no tumor) were each injectedintravenously with the 200 uL of the 750-pP-NPs. The bio-distribution of750-pP-NPs over time in each mouse were traced and quantified for2-hr-period with 10-min intervals. If the NP chaperones have been storedin the fridge for more than 6 hr, it is suggested to spin them down in100k filter column in a volume 20-fold of the original samples to getrid of the free peptides and/or VT-750.

The remaining pP-NPs from the nine selected samples were grouped intoseven MMP-specific pP-NPs (MMP:pP-NPs) and two thrombin-specific pP-NPs(thrombin:pP-NPs); the pP-NPs in each group were present in the sameamount based on peptide concentrations. 2 n-mol of MMP:pP-NPs (based onpeptide concentrations) was injected into each of the 16 nude mice (4with HT-1080 tumors, 4 with MDA-MB-435 tumors, 4 with no tumor butinjury, and 4 with no tumors and no injury); the urine of all 12 micewere collected after 2 hr of injections. 2 n-mol of thrombin:pP-NPs wereinjected into 8 Swiss Webster mice (immediately after injection, 4 wereslightly injured on both thigh muscles) and 4 nude mice with MDA-MB-435tumors; the urine of all 12 mice were collected after 1 hr ofinjections. In addition, 2 n-mol of a free thrombin peptide was injectedinto each of the 8 mice, and 4 were immediately injured afterward; theurine samples of all 8 mice were collected after 1 hr of injections. Inthe waiting periods between the injections of the NP chaperones and theexcretions of the urine, all mice were anesthetized with isoflurane(2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane). After urineexcretions for each mouse, each urine sample volume was diluted to 500uL by adding ddH₂O. 100 μL of each 500 uL urine sample was put into awell on a black half-96-well plate, and a microplate fluorimeter(Molecular Devices Corporation; Gemini EM; excitation: 485 nm, emission:538 nm, cutoff: 530 nm) was use to measure the relative fluorophoreunits (RFU) of each sample. Furthermore, 100 μL of each 250 uL urinesample was analyzed on HPLC-MS-MS for specific peptide sequencesinjected into the mice.

Statistical Analysis

Since that the sample sizes for both the nude mice and the white micewere very small and that a normal distribution could not be assumed, aStudent two-tailed t-test was performed for all statistical tests inthis study. The null hypothesis was that the two groups (e.g. tumor vs.non-tumor; injured vs. non-injured) do not differ in the amount ofpeptides excreted, and p-value <0.05 would reject the null hypothesis.

Example 2 Preparation of Pro-Diagnostic Reagents

43 pro-diagnostic reagents composed of peptide-PEG-nanoparticles(pP-NPs) were prepared and analyzed with a spectrometer to assess thenumber of peptides bound to each nanoparticle. The reagents weredesigned to enable rapid high throughput screening methods. FIG. 1 is aschematic depicting a method according to the invention for multiplexedin vivo enzyme profiling of mass-coded nanoparticle based pro-diagnosticreagents.

Example 3 In Vitro Screen of Extracellular Protease Activations onPro-Diagnostic Reagents

Initially, we established that the pro-diagnostic reagents could targettumors or injuries. We then conducted a screen of 43 pro-diagnosticreagents to fluorescently find optimal sequences for detection of tumorand injury proteases. The protease activities for a particular peptidesubstrate were measured by the fluorophore intensity levels over time.When the peptide-fluorophore (signature molecule) was attached to thenanoparticle iron core (carrier), the fluorescence of the fluorophorewas quenched by the neighboring absorption iron cores. However, when thepeptides were cleaved, the fluorophores were no longer quenched and, anincrease in fluorescence was detected.

FIG. 3A is a schematic of the pro-diagnostic reagent, with the circlesreferring to the carrier, the star is a signature molecule, and thezigzag line refers to the enzyme susceptible domain. FIG. 3B is a graphdepicting fluorescence activation versus time. FIG. 3C depicts data on43 pro-diagnostic reagents (with enzyme susceptible domains listed tothe right for detection of tumor and injury enzymes.

FIG. 4 is a Table depicting the mass detection of ejected fragments invitro. The results confirmed that the fluorescent results from thescreen could also be detected by analyzing the mass of ejected fragmentsin vitro.

Example 4 Tumor and Wound Targeting with Pro-Diagnostic Reagents

Tumor and wound targeting with pro-diagnostic reagents shows how thecarriers enhance the circulation time of the peptides (which wouldotherwise clear within minutes) and enable targeting to either tumors orinjuries. FIG. 2 shows data depicting the process of tumor and woundtargeting with pro-diagnostic reagents. FIG. 2A is a schematic of thepro-diagnostic reagent, with the circles referring to the carrier, thestar is a fluorescent molecule, and the zigzag line refers to the enzymesusceptible domain and the signature molecule (darker end region). FIG.2B is an electron micrograph of the pro-diagnostic reagent. FIG. 2C is agraph depicting the circulation time of the pro-diagnostic reagent, byplotting detection of the carrier in the blood with respect to timeafter intravenous injection. FIG. 2D is photographs of mice havingeither tumors or injuries (left and right panels, respectively)administered the pro-diagnostic reagent. FIG. 2E is histopathologicalanalysis of carrier homing to tumors or regions of injury.

Example 5 In Vivo Assessment of Tumor and Clotting-Factor ProteaseActivity

The circulation time of pro-diagnostic reagents in vivo was assessed.Optimized pro-diagnostic reagents were injected intravenously into miceand the cleaved peptide (signature component) was fluorescently trackedinto the urine after injection. The intensity of the fluorescence in theblood plasma sample was detected with the Odyssey imaging system. FIG. 5depicts the results of fluorescent detection of urinary reporteractivation by tumors and injuries in vivo. FIG. 5A is a schematic of thepro-diagnostic reagent as shown in FIG. 3A, further depicting theportion of the molecule that undergoes renal clearance and the portionthat undergoes RES clearance. FIG. 5B is a set of photographs of thatwere intravenously administered the optimized pro-diagnostic reagent forinjury detection (top) or tumor detection (bottom). Half the mice thatwere administered the optimized pro-diagnostic agents forinjury-detection suffered bilateral hind limb injuries (left side of thephotograph) while the control mice had no injuries (right side of thephotograph). Half the mice administered the optimized pro-diagnosticagents for tumor-detection harbored human fibrosarcoma tumors (HT-1080)(left side of photograph), while the other mice contained no tumors(right side of photograph). FIG. 5C is a set of graphs depictingrelative bladder fluorescence for tumor (bottom panel) or injured (toppanel) versus control mice in order to track the entrance of cleavedsignature peptide into the urine after injection. Bi-exponential linesof best fit were drawn for all samples to calculate the half-lifepoints.

FIG. 6 shows LC/MS quantitation of signature molecules in urine. FIG. 6Ais a photograph of an experimental mouse, having bilateral injury and acontrol uninjured mouse. FIG. 6B is a graph depicting the ratio ofsignature molecule (from thrombin cleavable proteolytic susceptibledomain) to isotopically labeled product in injured versus control mice.

Example 6 In Vitro Assessment of an Implantable Diagnostic Capsule

FIG. 7 shows an exemplary embodiment of an implantable diagnosticcapsule. FIG. 7A shows a photograph of a typical implantable capsule asdescribed, for example, in Daniel, K. D., et al., Implantable diagnosticdevice for cancer monitoring. Biosensors and Bioelectronics 24 (2009)3252-3257 which can be used according to the invention for loadingnanoparticles. Nanoparticles can be loaded into the well-shapedreservoir and sealed by a semi-permeable polycarbonate membrane enablingfree traffic of enzymes and other molecules but limiting theextracapsule diffusion of nanoparticles. FIG. 7B shows measurements ofthrombin-cleaved peptide efflux from implantable diagnostic capsulessealed with semi-permeable membranes of different pore sizes. Capsulesmade with membranes of pore size 10, 30, 50 or 80 nm were loaded withnanoparticles functionalized with GGdFPipRSGGGC (SEQ ID NO: 8) andexposed to solutions of thrombin or factor Xa (a cognate and anon-cognate protease, respectively). Thrombin-specific cleavage wasmonitored over time by measuring extracapsule fluorescence every 30minutes, normalizing over factor Xa. The kinetics of reporter release isshown.

Example 7 Optimization of Peptide Reporters for LC/MS Detection

A series of peptides (A1-A14) of different sequence were investigated todetermine optimal sequence length and charge density that would enablefacile detection via LC/MS. Three representative proteolytic products,sequences GGVVVLS (SEQ ID NO: 19), GGPVG (SEQ ID NO: 13), and GGdFPipR(SEQ ID NO: 17) were selected and appended with peptide caps ofdiffering length and charge density (FIG. 8A). The sequences were thendetected via LC/MS. FIG. 8B shows normalized relative intensities of thepeptide reporters. The inset of FIG. 8B shows a magnification of thenormalized relative intensities of the peptide sequences A1-A6 asmeasured via LC/MS. In general, hydrophilic sequences appended withshorter caps were more readily detected. For example, peptides A5 andA6, containing the shortest caps of the group of peptides containing theproteolytic product GGVVVLS (SEQ ID NO: 19), were more readily detectedthan other peptides of that group, which contained longer, or lesshydrophilic caps. A hydrophilic molecule is a molecule that cantransiently hydrogen-bond with water and is, thus, soluble in water and,in some embodiments, in other polar solvents. Hydrophilicity of peptidescan be modulated according to methods well known in the art. In someembodiments, removal of the fluorescein tag will increase thehydrophilicity of the peptides reported in this disclosure. In someembodiments, modification of the sequences to contain positively chargedresidues (Histidine, Lysine, and Arginine) and/or negatively chargedresidues (glutamic acid and aspartic acid) will increase thehydrophilicity. Similarly, peptides A11 and A12, containing the shortestcaps of the group of peptides containing the proteolytic product GGPVG(SEQ ID NO: 13), were more readily detected than other peptides of thatgroup, which contained longer, or less hydrophilic caps. Of the testedcap sequences Fl-dR-dS-dR (SEQ ID NO: 20) and Fl-dR-G-dS-dR (SEQ ID NO:21) were determined to be advantageous. Accordingly, for non-fluorescentdetection, the cap sequences dR-dS-dR (SEQ ID NO: 35) and dR-G-dS-dR(SEQ ID NO: 36) were determined to be advantageous. Using the resultsfrom the optimization experiments as guidelines, a revised list ofpro-diagnostic peptides were designed for optimal LC/MS detection. Thefollowing pro-diagnostic peptides optimized for LC/MS detection weredesigned:

Peptide Sequence A Fl-dR-dS-dR-G-G-P-Q-G-I-W-G-Q-C  (SEQ ID NO: 22) BFl-dR-G-dS-dR-G-G-P-L-G-V-R-G-K-C  (SEQ ID NO: 23) CFl-dR-G-dS-dR-G-G-P-L-A-Nva-Dpa-A-R-G-C  (SEQ ID NO: 24) DFl-dR-G-dS-dR-G-G-P-V-G-L-I-G-C  (SEQ ID NO: 25) EFl-dR-dS-dR-G-G-P-V-P-L-S-L-V-M-C  (SEQ ID NO: 26) FFl-dR-G-dS-dR-G-G-V-V-V-L-S-M-T-A-C  (SEQ ID NO: 27) GFl-dR-G-dS-dR-G-G-S-G-G-P-L-G-L-R-S-W-C  (SEQ ID NO: 28) HFl-dR-G-dS-dR-G-G-G-P-W-G-I-W-G-Q-G-C  (SEQ ID NO: 29) IFl-dR-G-G-dS-G-G-dF-Pip-R-S-G-G-G-C  (SEQ ID NO: 30) JFl-dR-dS-dR-G-G-L-V-P-R-G-S-G-C  (SEQ ID NO: 31) IaFl-dR-G-G-dS-G-G-F-P-R-S-G-G-G-C  (SEQ ID NO: 32) IbFl-dR-G-G-dS-G-G-G-dF-Pip-K-S-G-G-G-C  (SEQ ID NO: 33) IcFl-dR-G-G-dS-G-G-G-dF-P-K-S-G-G-G-C  (SEQ ID NO: 34)

Example 8 In Vitro Multiplexed Analysis of Protease Activity by LC/MS

Two identical cocktails of 12 pro-diagnostic nanoparticles, eachfunctionalized with a different peptide (FIG. 9A), were exposed tothrombin or collagenase and the proteolytically released reporters wereanalyzed by LC/MS. Six of the peptides contained target peptidesequences for thrombin, whereas the other six peptides contained targetpeptide sequences for collagenase. LC/MS peak area measurements of alltwelve peptides after exposure of the first multiplex cocktail tothrombin and after exposure of the second multiplex cocktail tocollagenase are shown in FIG. 9B and FIG. 9C, respectively. In thecocktail exposed to thrombin, peptide reporters cleaved from peptidescontaining a thrombin target site (left six bars) were predominantlydetected over peptide reporters cleaved from peptides containing acollagenase target site (right six bars), as shown in FIG. 9B. In thecocktail exposed to collagenase, peptide reporters cleaved from peptidescontaining a collagenase target site (right six bars) were predominantlydetected over peptide reporters cleaved from peptides containing athrombin target site (left six bars), as shown in FIG. 9C. FIG. 9D showsthe ratio of the LC/MS peak area for each peptide reporter afterexposure to thrombin over the peak area measured after exposure tocollagenase.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not limited in scope by the examples provided, since theexamples are intended as illustrations of various aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

The invention claimed is:
 1. A method comprising: introducing adiagnostic reagent, wherein said diagnostic reagent comprises a carrierlinked to a signature molecule by a peptide, wherein said peptide iscleaved by a protease, and wherein said diagnostic reagent is notcoupled to a chip; detecting said signature molecule cleaved from saidcarrier; and administering to said subject an effective amount of apharmaceutical composition comprising a therapeutic agent.
 2. The methodof claim 1, wherein said signature molecule comprises a peptidesequence, a nucleic acid, a small molecule, a fluorophore, acarbohydrate, a particle, a radiolabel, a MRI-active compound, aninorganic material, and/or an organic material.
 3. The method of claim2, wherein said diagnostic reagent further comprises a quencherconfigured to quench the fluorophore.
 4. The method of claim 1, whereinsaid cleavage of said peptide generates a detectable signal.
 5. Themethod of claim 1, wherein said protease cleaves said peptide in vivo.6. The method of claim 1, wherein said protease cleaves said peptide invitro.
 7. The method of claim 1, wherein said protease is a tumorprotease.
 8. The method of claim 1, wherein said protease comprisesMMP-2, MMP-7, MMP-8, MMP-9, MMP-14, thrombin, factor Xa, tissue factor,cathepsin B, or a combination thereof.
 9. The method of claim 1, whereinsaid carrier comprises a particle, further wherein said particle is amicroparticle or a nanoparticle.
 10. A method comprising: introducing adiagnostic reagent, wherein said diagnostic reagent comprises a carrierlinked to a signature molecule by a peptide, wherein said peptide iscleaved by a protease, and wherein said diagnostic reagent is notcoupled to a chip; performing an assay to detect said signature moleculecleaved from said carrier; and evaluating a disease based on aquantifiable amount of said signature molecule, wherein evaluating saiddisease is based on an amount of said cleaved signature molecule, andwherein said disease is a liver disease.
 11. The method of claim 10,wherein said signature molecule comprises a peptide sequence, a nucleicacid, a small molecule, a fluorophore, a carbohydrate, a particle, aradiolabel, a MRI-active compound, an inorganic material, and/or anorganic material.
 12. The method of claim 11, wherein said diagnosticreagent further comprises a quencher configured to quench thefluorophore.
 13. The method of claim 10, wherein said cleavage of saidpeptide generates a detectable signal.
 14. The method of claim 10,wherein said protease cleaves said peptide in vivo.
 15. The method ofclaim 10, wherein said protease cleaves said peptide in vitro.
 16. Themethod of claim 10, wherein said protease comprises MMP-2, MMP-7, MMP-8,MMP-9, MMP-14, thrombin, factor Xa, tissue factor, cathepsin B, or acombination thereof.
 17. The method of claim 1, wherein said therapeuticagent comprises releasable drugs.